Methods of administering dalbavancin for treatment of bacterial infections

ABSTRACT

The invention provides methods and compositions for treatment of bacterial infections. Methods of the invention include administration of dalbavancin for treatment of a bacterial infection, in particular a Gram-positive bacterial infection of skin and soft tissue. Dosing regimes include once weekly administration of dalbavancin, which often remains at therapeutic levels in the bloodstream for at least one week, providing prolonged therapeutic action against a bacterial infection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/829,068, filed Apr. 20, 2004, which is a continuation of U.S.application Ser. No. 10/714,261, filed Nov. 14, 2003, now U.S. Pat. No.6,900,175, which claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 60/427,654, filed Nov. 18, 2002, 60/485,694, filedJul. 8, 2003, 60/495,048, filed Aug. 13, 2003, and 60/496,483, filedAug. 19, 2003, the disclosure of all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This application relates to dalbavancin compositions and methods of useof such compositions in methods of treatment of bacterial infections.

BACKGROUND OF THE INVENTION

According to the U.S. Center for Disease Control and Prevention,nosocomial bloodstream infections are a leading cause of death in theUnited States. Approximately five percent of the seven million centralvenous catheters (CVCs) inserted annually in the United States areassociated with at least one episode of bloodstream infection(approximately 350,000 a year). Catheter-related bloodstream infectionsoccur when bacteria enter the bloodstream through an intravenouscatheter and can be life threatening.

Skin and soft tissue infections (SSTIs) are a common medical conditionand often the consequence of trauma or surgical procedures.Staphylococcus aureus and Streptococcus pyogenes are the pathogens mostfrequently isolated from patients with deep tissue infections, althoughany pathogenic organism, including those found on healthy skin, maycause infection. Many SSTIs are mild to moderate in severity, permittingsuccessful treatment with oral antimicrobial agents and local cleansing.In contrast, more severe or complicated infections, which frequentlyoccur in patients with underlying risk factors (e.g., vascularcompromise, diabetes) and/or infections caused by difficult-to-treat ormultiply-resistant bacteria, may require potent intravenousantimicrobial therapy and aggressive surgical debridement.

Staphylococci are a clinical and therapeutic problem and have beenincreasingly associated with nosocomial infections since the early1960s. The coagulase-positive species methicillin resistantStaphylococcus aureus (MRSA) has long been problematic in bothcommunity-acquired and nosocomial infections, and severalcoagulase-negative staphylococci have been recognized as opportunistichuman pathogens, especially in the treatment of critically ill patientsin intensive care units. Another major cause for clinical concern is theincreasing isolation of penicillin-resistant Streptococcus pneumoniaestrains in many parts of the world.

The glycopeptide antibiotics vancomycin and teicoplanin have been usedagainst serious nosocomial infections caused by multi-drug-resistantGram-positive pathogens, particularly MRSA, coagulase-negativestaphylococci (CoNS), and enterococci. Vancomycin and teicoplanin areused for infections caused by MRSA, and until recently, all isolateswere uniformly susceptible. However, the isolation of Staphylococcusaureus strains with intermediate susceptibility or resistance toteicoplanin as well as vancomycin has now been reported with increasingfrequency. A number of vancomycin-resistant strains, classified “VanA,”“VanB,” or “VanC,” based on the mechanism of resistance, have beenreported. Thus, alternative treatment options are needed.

Teicoplanin is at least as active as vancomycin against mostGram-positive bacteria and appears to cause fewer adverse events. Bothforms of treatment require at least once daily dosing to effect completerecovery. Currently, the therapeutic options for severe infectionscaused by some of these pathogens is quite limited. The emergingresistance of Gram-positive pathogens to vancomycin makes theavailability of new antibiotics with potential for increasedeffectiveness highly desirable.

In addition, less frequent dosing regimens than currently-availabletherapies would be desirable to enhance patient comfort, especially forparenteral, e.g., intravenous or intramuscular, antibioticadministration. Hospital stays are sometimes necessitated by the needfor multi-daily antibiotic administration by parenteral means, and lessfrequent dosing would be advantageous to permit such treatment to bedone on an outpatient basis.

Although less frequent dosing is a desirable feature of an antibioticadministration regimen, the “pharmaceutical window,” i.e., the toxicityprofile, of the administered antibiotic must be sufficiently acceptableto permit a large single dose to be administered without jeopardizingtreatment by causing severe adverse reactions in the treated patient.Further, even when an antibiotic exhibits a suitable pharmaceuticalwindow, less frequent dosing is possible only if the antibiotic exhibitsa suitable serum half-life to maintain therapeutic effectiveness overthe dosing interval desired. The serum half-life of an antibioticdictates both the longevity of a drug in vivo and the length of timeafter administration when the serum level will reach a minimum troughlevel which is still bactericidally effective. The serum trough levelover time after administration of a first dose of antibiotic dictateswhen a further dose must be administered to retain a minimumbactericidal level of the antibiotic in vivo.

In view of the above, an antibiotic possessing activity against one ormore antibiotic resistant bacterial strains, particularly MRSA, whichcould be administered at a dosing interval of once every 5-7 days orlonger, would be of commercial value and would satisfy a long-felt needin the art.

SUMMARY OF THE INVENTION

The invention provides compositions, methods and kits for treatment orprevention of a bacterial infection with dalbavancin. Surprisingly,stabilized formulations of dalbavancin have been found to exhibit both apharmaceutical window as well as a prolonged serum half-life to permittreatment regimens of about once every 5-7 days or longer, whileretaining antibacterial properties in vivo.

Accordingly, in one aspect, a pharmaceutical composition is providedthat includes a unit dose of dalbavancin in an amount sufficient toprovide a therapeutically or prophylactically effective plasma level ofdalbavancin in an individual for at least five days, a stabilizer, and apharmaceutically acceptable carrier.

Pharmaceutical compositions of the invention are generally formulated ina pharmaceutically acceptable form for administration to an individual,such as a pharmaceutically acceptable aqueous formulation. Suchpharmaceutical compositions are preferably administered by parenteral,e.g., intravenous or intramuscular, routes. Accordingly, in thispreferred embodiment, these pharmaceutical compositions are typicallysterile.

In some embodiments, a unit dose of dalbavancin is provided in drypowder (e.g., lyophilized) form and reconstituted in a pharmaceuticallyacceptable carrier, such as a sterile aqueous formulation, prior toadministration to an individual. In one embodiment, the pharmaceuticallyacceptable carrier includes 5% dextrose in water. A pharmaceuticalcomposition of the invention may be administered to a mammal in need oftreatment or prevention of a bacterial infection, such as a human. Insome embodiments, a pharmaceutical composition may include at least oneantibiotic that is not dalbavancin, such as an antibiotic that iseffective (e.g., bactericidal) against a Gram-negative bacterium and/oran antibiotic that is effective against Gram-positive species againstwhich dalbavancin is not effective, such as VanA vancomycin-resistantbacterial strains.

One or more stabilizing substances are employed to inhibit degradationof one or more dalbavancin components during storage as a dry powder(e.g., lyophilized) formulation and/or as an aqueous formulation priorto administration to an individual. Over time, degradation can result inthe undesirable formation of less active and/or inactive componentswhich could potentially cause adverse effects in vivo. Preferredstabilizers include nonionic components such as sugars or sugaralcohols, e.g., a mono-, di-, or polysaccharide, or derivative thereof,such as, for example, mannitol, lactose, sucrose, sorbitol, glycerol,cellulose, trehalose, maltose, or dextrose, or mixtures thereof.

In another aspect, methods are provided for treating a bacterialinfection in an individual in need thereof, including administering atleast one unit dose of dalbavancin in an amount sufficient to provide atherapeutically effective plasma level of dalbavancin in the individualfor at least five days, and a pharmaceutically acceptable carrier. Atherapeutically effective plasma level of dalbavancin is generally atleast about 4 mg of dalbavancin per liter of plasma. In one embodiment,the dosage amount of dalbavancin administered is an amount that isclinically effective and also has reduced adverse side effects incomparison to the standard of care with drugs such as teicoplanin andvancomycin.

Dalbavancin may be administered as a single dose or as multiple doses.In some embodiments, a single dose of about 100 mg to about 4000 mg, forexample 3000 mg, of dalbavancin is administered. In various embodiments,a single dalbavancin dose may include at least about any of 0.1, 0.25,0.5, 1, 1.5, 2, 2.5, or 3 grams.

In other embodiments, two doses are administered about five to about tendays apart, such as about one week apart. The first dose may be about500 to about 5000 mg of dalbavancin and the second dose may be about 250mg to about 2500 mg of dalbavancin. Often, the first dose includes about1.5 to about 3 times, often at least about twice as much of the amountof dalbavancin contained in the second dose. For example, the first dosemay be about 1000 mg and the second dose may be about 500 mg ofdalbavancin. In methods in which two doses are administered, the plasmatrough level of dalbavancin in an individual prior to administration ofthe second dose is generally at least about 4 mg, often at least about10 mg, often at least about 20 mg, more often at least about 30 mgdalbavancin per liter of plasma, and still more often at least about 40mg dalbavancin per liter of plasma.

Often, methods of the invention include parenteral administration, forexample intravenous administration. In some embodiments, administrationis intravenous with the rate of administration controlled such thatadministration occurs over at least about 30 minutes or longer.

Methods of the invention may be used to treat a Gram-positive bacterialinfection, such as, for example, a Staphylococcus aureus orStreptococcus pyogenes skin and soft tissue infection. In someembodiments, the infection is penicillin-resistant and/or multi-drugresistant.

In another aspect, a method for preventing a bacterial infection isprovided which includes administering at least one unit dose ofdalbavancin in an amount sufficient to provide a prophylacticallyeffective plasma level of dalbavancin in the individual for at leastabout one day, three days, five days, one week, or ten days or longer,and a pharmaceutically acceptable carrier. The dosage of dalbavancin maybe, for example, about 100 mg to about 1000 mg. In some embodiments,dalbavancin is administered prior, during, or subsequent to a medicalprocedure or a stay in the hospital.

Therapeutic or prophylactic methods of the invention may includeadministration of at least one antibiotic that is not dalbavancin,preferably an antibiotic that is effective against a Gram-negativebacterium and/or an antibiotic that is effective against Gram-positivestrains that dalbavancin is not effective against, such as VanA strains.

In another aspect, kits are provided that include at least one unit doseof dalbavancin in an amount sufficient to provide a therapeuticallyeffective plasma level for at least about five days or aprophylactically effective plasma level of dalbavancin for at leastabout one day in an individual, and instructions for use in a method oftreatment or prophylaxis of a bacterial infection. A kit may contain twounit dosages, with a first dosage including 1.5 to 3 times, often atleast about twice as much of the amount of dalbavancin included in asecond dosage. Kits may also include an antibiotic that is notdalbavancin, preferably effective against a Gram-negative bacterium.

In one embodiment, kits are provided that include a first containercontaining a dry powder (e.g., lyophilized) dalbavancin composition anda second container containing a predetermined amount of aphysiologically acceptable aqueous solution for admixing with thedalbavancin composition. Such solutions are preferably sterile aqueoussolutions. In one embodiment, kits include a delivery means foradministering the dalbavancin composition to an individual, for examplea syringe or intravenous administration means.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts dalbavancin plasma concentration versus time following asingle 1000 mg intravenous infusion of dalbavancin.

FIG. 2 depicts isothermal titration calorimetry data for dalbavancinbinding to human serum albumin (top) and a graphical representation ofthe data fitted to a curve determined from a 2:1 binding model ofdalbavancin:protein (bottom).

FIG. 3 depicts an electrospray ionization mass spectrum of dalbavancin.

FIG. 4 is a graph of dalbavancin concentration vs. population ratio ofdalbavancin multimer to monomer and depicts an increase in populationratio of dalbavancin multimer to monomer with increasing dalbavancinconcentration.

FIG. 5 is a graph of pH vs. population ratio of dalbavancin multimer tomonomer and depicts an increase in population ratio of dalbavancinmultimer to monomer with increasing pH.

FIG. 6 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 5 mM pH 5 solution.

FIG. 7 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 50 mM pH 5 solution.

FIG. 8 depicts an electrospray ionization mass spectrum of dalbavancinin an ammonium formate 100 mM pH 5 solution.

FIG. 9 depicts an electrospray ionization mass spectrum of teicoplanin(50 μg/mL) in water.

FIG. 10 depicts an electrospray ionization mass spectrum of teicoplanin(100 μg/mL) in water.

FIG. 11 depicts the effect of HSA on the apparent dissociation constantfor dalbavancin/tri-peptide binding at 26° C. (pH 7.4).

FIG. 12 depicts a comparison of isothermal calorimetry (ITC) data forbinding of tri-peptide to vancomycin and dalbavancin under identicalconditions, using the same tri-peptide solution.

FIGS. 13A and 13B depict the possible interaction of dalbavancinmonomers and multimers (including dimers) with tri-peptide ligand andHSA. FIG. 13A depicts dalbavancin in monomer-dimer equilibrium insolution, binding as monomer to two separate sites on HSA. FIG. 13Bdepicts ligand binding to dalbavancin dimer in solution and more weaklyto dalbavancin monomers attached to HSA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved dosage regimes and novelcompositions of dalbavancin, and improved methods of treatment ofantibiotic-resistant bacterial infections. In particular, the inventionprovides dalbavancin compositions having activity against one or moreantibiotic resistant strains of bacteria, such as MRSA, which may beadministered in a dosing regimen of once every 5-7 days or longer.

Dalbavancin, which is also referred to in the scientific literature asBI 397 or VER001, is a semi-synthetic glycopeptide mixture, theproperties of which have been reported in U.S. Pat. Nos. 5,606,036,5,750,509, 5,843,679, and 5,935,238.

As used herein, the term “dalbavancin” refers to compositions comprisingone or more, preferably two or more, closely related homologs, termed“A₀,” “A₁,” “B₀,” “B₁,” “C₀,” and “C₁,” as described below, or monomers,multimers (i.e., dimer or higher order multimer), tautomers, esters,solvates, or pharmaceutically acceptable salts thereof. As used herein,“dimer” or “multimer” refers to either a homodimer or homomultimer,i.e., a dimer or multimer composed of monomers of the same dalbavancinhomolog, or a heterodimer or heteromultimer, i.e., a dimer or multimercomposed of monomers of at least two different dalbavancin homologs.Dalbavancin often includes “MAG,” a non-homolog variant described below.Individually, dalbavancin homologs and MAG are sometimes referred toherein as “dalbavancin components.”

Dalbavancin is prepared by chemical modification of the naturalglycopeptide complex A-40,926 as described in Malabarba and Donadio(1999) Drugs of the Future 24(8):839-846. The predominant component ofdalbavancin is Factor B₀, which accounts for >75% of the whole complex.

The amount of each of the components present in a dalbavancincomposition is dictated by a variety of factors, including, for example,the fermentation conditions employed in the preparation of the naturalglycopeptide complex A-40926, which is the precursor to dalbavancin(see, e.g., U.S. Pat. No. 5,843,679), the conditions employed to recoverA-40926 from the fermentation broth, the chemical reactions employed toselectively esterify the carboxyl group of the sugar moiety of A-40926,the conditions employed to amidate the peptidyl carboxyl group, theconditions employed to saponify the ester of the carboxyl group of theN-acylaminoglucuronic acid function, the conditions employed to recoverdalbavancin from the synthetic mixture, and the like.

In preferred embodiments, dalbavancin compositions comprise at leastabout 80 to about 98% by weight of the B₀ component. In particularlypreferred embodiments, dalbavancin comprises the following amounts ofB₀:

TABLE 1 Preferred Amounts of B₀ Component in Dalbavancin CompositionPreferred¹ More Preferred¹ Even More Preferred¹ 80-98 80-97 80-96 81-9881-97 81-96 82-98 82-97 82-96 83-98 83-97 83-96 84-98 84-97 84-96 85-9885-97 85-96 86-98 86-97 86-96 87-98 87-97 87-96 88-98 88-97 88-96 89-9889-97 89-96 90-98 90-97 90-96 ¹each range represents the mole % of B₀relative to the total dalbavancin components present in the dalbavancincomposition including MAG

The chemical structure of several of the dalbavancin components isdepicted in Formula I below:

I

Delbavancin Component R Molecular Weight A₀ —CH(CH₃)₂ 1802.7 A₁—CH₂CH₂CH₃ 1802.7 B₀ —CH₂CH(CH₃)₂ 1816.7 B₁ —CH₂CH₂CH₂CH₃ 1816.7 C₀—CH₂CH₂CH(CH₃)₂ 1830.7 C₁ —CH₂CH₂CH₂CH₂CH₃ 1830.7

All of the above dalbavancin components are bactericidally activeagainst a number of Gram-positive bacteria. However, one non-homologousdalbavancin component, termed “MAG,” which lacks an acylglucoronaminemoiety present in other components, is less bactericidally effective,both in vivo and in vitro, than other dalbavancin components. MAG isthought to be a decomposition product of one or more of the otherdalbavancin components. Accordingly, in a preferred embodiment, theamount of MAG in dalbavancin is less than about 4, 3.5, 3, 2.5, 2, 1.5,1, or 0.5 mole percent of all dalbavancin components present, includingMAG.

Dalbavancin is thought to inhibit the biosynthesis of the bacterial cellwall by binding to D-alanyl-D-alanine-terminating precursors ofpeptidoglycans. Dimeric or higher order multimers of dalbavancin maypossess further antibacterial properties by interaction of thelipophilic side chains with the cytoplasmic membrane of bacteria. See,for example, Malabarba and Ciabatti, et al. (2001) Current MedicinalChemistry 8:1759-1773. A further elaboration on dalbavancin multimersmay be found in U.S. Ser. No. 10/714,166, entitled “DALBAVANCINCOMPOSITIONS FOR TREATMENT OF BACTERIAL INFECTIONS,” filed on Nov. 14,2003, as Attorney Docket No. 34231-20052.00, the disclosure of which ishereby incorporated by reference in its entirety.

In vitro, nonclinical, and clinical data indicate dalbavancin to be ofbenefit for the treatment of serious Gram-positive infections caused byMRSA and CoNS, and all streptococcal and non-VanA enterococcal species,including VanB and VanC phenotypes poorly susceptible or resistant tovancomycin.

Dalbavancin is more active in vitro against staphylococci (includingsome teicoplanin-resistant strains) than teicoplanin and vancomycin.Dalbavancin has better activity against streptococci, includingpenicillin-resistant strains, than teicoplanin or vancomycin.Dalbavancin is active in vitro and in vivo against a number ofGram-positive bacteria, including most drug resistant strains.

Dalbavancin is typically administered to an individual as a dalbavancincomposition. As used herein, the term “dalbavancin composition” or“dalbavancin formulation” refers to a composition, typically apharmaceutical composition comprising dalbavancin, as defined above, andone or more other non-dalbavancin components such as, for example, apharmaceutically acceptable carrier, a stabilizer, a buffers, or othersimilar components.

As shown in Example 1, dalbavancin is effective at dose intervals of oneweek. Thus, an advantage of dalbavancin versus other treatment optionsis the ability to administer this antibiotic on a once-weekly basis,thereby maximizing patient compliance and potentially minimizing theneed for or decreasing the length of a hospital stay for parenteralantibiotic administration. Less frequent dosing often permits treatmenton an out-patient basis, thus decreasing treatment costs. As furthershown in Example 1, a second dose of dalbavancin approximately one weekafter administration of the first dosage, where the second dose isapproximately one-half the first dose, unexpectedly provides significantimprovement in the efficacy of treatment.

Methods of Use

Methods are provided for administration of dalbavancin to an individualin need of treatment for a bacterial infection. Treatment can includeprophylaxis, therapy, or cure. Methods include administration of one ormore unit doses of dalbavancin in a therapeutically or prophylacticallyeffective amount.

As used herein, “therapeutically effective amount” refers to the amountof dalbavancin that will render a desired therapeutic outcome (e.g.,reduction or elimination of a bacterial infection). A therapeuticallyeffective amount may be administered in one or more doses. A“prophylactically effective amount” refers to an amount of dalbavancinsufficient to prevent or reduce severity of a future bacterial infectionwhen administered to an individual who is susceptible to and/or who maycontract a bacterial infection, e.g., by virtue of a medical procedureor stay in the hospital, or exposure to an individual with a bacterialinfection. Dalbavancin is generally administered in a pharmaceuticallyacceptable carrier.

Dalbavancin is often provided as a hydrochloride salt, which is freelysoluble in water.

Typically, dalbavancin is administered as a “unit dose” in a dalbavancinformulation which includes an amount of dalbavancin sufficient toprovide a therapeutically or prophylactically effective plasma level ofdalbavancin for several days, often at least about 5 days, one week, or10 days, when administered to an individual.

As used herein, “individual” refers to a vertebrate, typically a mammal,often a human.

All homologs of dalbavancin described above exhibit a prolongedhalf-life in plasma, often 9 days or more, although MAG is thought tohave a shorter half-life than other homologs. The long half-life permitslonger intervals between dosages than vancomycin or teicoplanin. Asdescribed in Example 1, weekly dosing of dalbavancin is effective forcontrol of bacterial infections, in contrast to the twice daily dosingschedule which is often used for vancomycin or the once daily schedulegenerally used for teicoplanin. Less frequent dosing of dalbavancinoffers significant treatment advantages over vancomycin and teicoplanin,particularly with regard to improved convenience and patient compliancewith the treatment regimen. Surprisingly high doses (i.e., resulting insurprising high and long-lasting serum levels) can be administered, andwith less frequency than other available treatment options. The noveldosage regimen available for dalbavancin results in improved efficacybecause at concentrations required to effect less frequent dosing,dalbavancin exhibits minimal adverse effects in vivo, evidencing a largepharmaceutical window, and further because blood levels of dalbavancinare maintained above minimum bactericidal levels for the entiretreatment protocol, evidencing a prolonged serum half-life fordalbavancin. The combination of the large pharmaceutical window coupledwith prolonged serum half-life permits less frequent dosing ofdalbavancin.

In addition, dalbavancin is preferably formulated with a stabilizerwhich inhibits degradation of one or more of the components ofdalbavancin. In one preferred embodiment, dalbavancin is formulated witha 1:2 weight ratio of mannitol:dalbavancin. In another preferredembodiment, dalbavancin is formulated with a 1:1:4 weight ratio ofmannitol:lactose:dalbavancin.

In some embodiments, a dalbavancin formulation is administered at adosage that results in therapeutically effective (ice., bactericidal)plasma levels of the drug for several days, often at least about 5 toabout 10 days, often at least about one week. Generally, dalbavancin ismaintained in plasma at or above the minimum bactericidal concentrationof about 4 mg/l for at least 5 days. Often, dalbavancin is maintained ata plasma level of at least about 5 mg/l, often at least about 10 mg/l,often at least about 20 mg/l, often at least about 30 mg/l, often atleast about 40 mg/l, for at least 5 days, often at least about one weekor longer. Plasma levels of dalbavancin may be measured by methods thatare well known in the art, such as liquid chromatography, massspectrometry, or microbiological bioassay. An example of a method forquantitating dalbavancin in plasma is provided in Example 5.

Upper limits for dalbavancin plasma concentration levels are generallydictated by dosages which inhibit unacceptable adverse effects in thepatient population treated.

Dalbavancin compositions may be administered in a single dose or inmultiple doses. When administered as a single dose, the dalbavancincomposition is preferably formulated to contain sufficient amounts ofdalbavancin to effect antibacterial properties in vivo for at least 5days, preferably at least 7 days, and more preferably at least 10 days.

When multiple doses are employed, dalbavancin can be administered weeklyfor two or more weeks. In one embodiment, dalbavancin is administered inat least two doses, often in two doses about 5 to about 10 days apart,more often once a week for two weeks. As shown in Example 1, such adosing regimen provides significant advantages over conventionalantibiotic treatment protocols.

Dalbavancin compositions also may be administered in multiple doses twoor more days or at least one week apart or in one or more biweeklydoses. In some embodiments, a dalbavancin composition is administeredweekly, followed by biweekly, or monthly administration. In someembodiments, dalbavancin is administered at weekly intervals for 2, 3,4, 5, 6, or more weeks.

Most advantageously, daily dosing is not required because higher, lessfrequent doses are used. Single or multiple doses may range, forexample, from about 0.1 to about 5 grams. A single dose of about 0.1 toabout 4 grams, e.g., about 3 grams, may be administered for variousinfection treatments. Where multiple doses are administered, forexample, weekly, each dose may range, for example, from about 0.25 toabout 5.0 grams.

For embodiments in which a single dose is administered to treat aninfection, the amount of the dose may be, for example, about 0.1 toabout 5 grams, or about 0.5 to about 4 grams, or about 1 to about 3.5grams, or about 2 to about 3 grams e.g., about 3 grams. In someembodiments, a single dose of about 1, 1.5, 2, 2.5, or 3 grams isadministered for treatment of a bacterial infection. For embodiments inwhich a single dose is administered for prophylaxis, the amount of thedose may be, for example, about 0.1 to about 3 grams, or about 0.1 toabout 1 gram, e.g., about 0.5 or about 0.25 gram.

In dosing schemes that include multiple dosages, the individual dosagesmay be the same or different. In some embodiments, a first, higher doseis administered, that is, for example, about 1.5 to 3 times higher, thanone or more subsequent doses. For example, the first dose may be about0.5 grams to about 5 grams and the second dose about 0.25 grams to about2.5 grams, the first dose may be about 0.8 to about 2 g and the seconddose about 0.4 to about 1 gram, or the first dose may be about 0.4 toabout 3 g and the second dose about 0.2 to 1.5 g.

In some embodiments, at least two dosages are administered wherein thefirst dosage includes about twice as much dalbavancin as subsequentdosages. In one embodiment, a first dosage includes about 1 gram ofdalbavancin and a subsequent dosage includes about 0.5 gram. In anotherembodiment, a first dosage includes about 0.5 gram of dalbavancin and asubsequent dosage includes about 0.25 gram.

In some embodiments, a dalbavancin composition is administered in twodoses of equal or different amount two or more days or at least aboutone week apart. Often, two doses of about 0.2 to about 1.5 grams ofdalbavancin are administered about 5 to about 10 days apart, more oftenabout 1 week apart. In one embodiment, a first dosage of about 1 gram ofdalbavancin and a second dosage of about 0.5 gram of dalbavancin areadministered about 1 week apart.

In a multiple dosing regimen, the time between doses may range, forexample, from about 5 to about 10 days, often about one week. Dosefrequency may be, for example, two weekly doses, or multiple weeklydoses. The dosing interval, or time between doses, can be, for example,any of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or moredays. The number of doses given, can be, for example, one, two, three,four, five, six or more doses, each dose after the initial dose beinggiven after the selected dosage interval.

In a multiple dosing scheme, often the “trough level,” or the level ofdalbavancin in plasma after a first dose of dalbavancin and just priorto administration of a second dose, is at least about 4 mg/l.Preferably, the trough level at the end of a dosing interval such asabout one week is at least about 20 mg/l, more preferably at least about30 mg/l, and even more preferably at least about 40 mg/l.

Dalbavancin can be administered parenterally, e.g., intramuscularly(i.m.), intravenously (i.v.), subcutaneously (s.c.), intraperitoneally(i.p.), or intrathecally (i.t.). The dosing schedule and actual dosageadministered may vary depending on such factors as the nature andseverity of the infection, the age, weight, and general health of thepatient and the tolerance of a particular patient to dalbavancin, butwill be ascertainable to health professionals. In one embodiment, a onegram intravenous dose of dalbavancin is followed by a 0.5 gramintravenous dose one week later.

Administration and delivery of the drug to the patient, e.g.,intravenously, can be done at a controlled rate, so that theconcentration in the blood does not increase too quickly or causeprecipitation to occur. In some embodiments, dalbavancin is administeredat an appropriate rate such that the drug forms a complex withendogenous protein(s) in the bloodstream. Without intending to be boundto a particular theory, it is believed that endogenous protein, such ashuman serum albumin, can form a complex in vivo with one or twomolecules of dalbavancin homolog monomers. When a sufficient amount ofdalbavancin is present, it is believed that up to two molecules ofdalbavancin homolog will bind to the endogenous protein and it isfurther believed that this complex is formed by binding of separatehomolog molecules of dalbavancin at two different binding sites.Alternatively, it is possible that dimeric dalbavancin is binding to asingle binding site on the endogenous protein. A further elaboration onthe dalbavancin-endogenous protein complexes discussed above may befound in U.S. Ser. No. 10/713,924, entitled “COMPOSITIONS AND METHODSFOR TREATING BACTERIAL INFECTIONS WITH PROTEIN-DALBAVANCIN COMPLEXES,”filed on Nov. 14, 2003 as Attorney Docket No. 34231-20053.00, thedisclosure of which is hereby incorporated by reference in its entirety.

The infusion duration can be, for example, about 1 minute to about 2hours. For example, an infusion duration of about 30 minutes may be usedwhere the dose is about 0.5 to about 1 gram. Intravenous administrationunder controlled rate conditions can generate concentrations ofdalbavancin in the body that are in great excess of what can be achievedin the solution phase at physiological pH in vitro. Although not wishingto be limited by theory, this may be due to the formation of a complexof dalbavancin with endogenous protein(s) such as serum albumin, whichmay increase the capacity of plasma to absorb dalbavancin.

Formation of a dalbavancin complex in vitro or ex vivo may permit fasteradministration, such as at least about 1 minute, at least about 10minutes or at least about 20 minutes. Such a complex can be achieved bymixing human serum albumin and/or another endogenous protein withdalbavancin, thereby forming the complex in vitro or ex vivo, and thenadministering this complex to the treated patient. Alternatively, thehuman serum albumin or other endogenous protein may be obtained fromautologous sources or by expression from a microorganism modified tocontain the gene for the protein.

The amount of dalbavancin administered may be any of the dosagesdisclosed herein. The dalbavancin dose is generally chosen such that thedrug will remain at a therapeutically or prophylactically effective(i.e., bactericidal) plasma level for an extended period of time, oftenat least 5 days, more often about one week or longer. Administration ofa dose of dalbavancin which produces and maintains bactericidalconcentrations for at least about one week (or about 5 to about 10 days)is preferred. A bactericidal concentration is defined as theconcentration of dalbavancin required to kill at least 99% of thebacteria present at the initiation of an in vitro experiment over a 24hour period. A minimum bactericidal concentration of dalbavancin inplasma is typically about 4 mg/l.

Examples of indications that can be treated include both complicated anduncomplicated skin and soft tissue infections (SSTI), blood streaminfections (BSI), catheter-related blood stream infections (CRBSI),osteomyelitis, prosthetic joint infections, surgical prophylaxis,endocarditis, hospital or community acquired pneumonia, pneumococcalpneumonia, empiric treatment of febrile neutropenia, joint spaceinfections, and device infections (e.g., pace makers and internalcardiac defibrillators). Gram-positive or antibiotic-resistant bacterialinfections may be treated, such as a Staphylococcus, Streptococcus,Neisseria, or Clostridium genus infection, in particular Staphylococcusaureus, Staphylococcus epidennidis, Staphylococcus hemolyticus,Streptococcus pyogenes, Groups A and C Streptococcus, Neisseriagonorrhoeae, or Clostridium difficile.

The invention provides methods for treatment of skin and soft tissueinfections (SSTIs). Patients who may benefit from this treatment mayhave either deep or superficial infections. SSTI may involve deeper softtissue and/or require significant surgical intervention, such as forexample a major abscess, infected ulcer, major burn, or deep andextensive cellulitis. Infected surgical wounds may also be treated.

The clinical presentation of skin and skin structure infection may varyfrom mild folliculitis to severe necrotizing fasciitis. The mode ofacquisition may also vary with community-acquired skin and skinstructure infections, which are often preceded by injuries resultingfrom occupational exposure or recreational activities, and are usuallyassociated with a greater diversity of pathogens. Hospital-acquired skinand skin structure infections are generally associated with surgicalprocedures, the development of pressure sores, and catheterization.Post-surgical infections are the third most frequent nosocomialinfection and account for 17% of all nosocomial infections reported tothe National Nosocomial Infection Surveillance System (NNIS). The mostfrequent source of infection is the patient's endogenous flora.Staphylococcus aureus, coagulase-negative staphylococci, andEnterococcus spp. are the pathogens most frequently isolated from SSTIs.

Symptoms of SSTI infections may include erythema, tenderness or pain,heat or localized warmth, drainage or discharge, swelling or induration,redness, or fluctuance. Patients that may benefit from treatment withthe methods of the invention include those with deep or complicatedinfections or infections that require surgical intervention, or patientswith underlying diabetes mellitus or peripheral vascular disease. Theseinfections are often caused by Gram-positive bacteria such asStaphylococcus or Streptococcus species, such as Staphylococcus aureusor Streptococcus pyogenes. Methods for treatment of a skin or softtissue bacterial infection include administering a therapeuticallyeffective amount of dalbavancin to an individual in need of treatment,in an amount and according to a dosing regime as discussed above. Insome embodiments, a dalbavancin composition is administeredintravenously in two doses, often about 5 to about 10 days apart, moreoften about 1 week apart. In some embodiments, the first dosage includesat least twice as much dalbavancin as the second dosage. In oneembodiment, the first dosage is about 1000 mg and the second dosage isabout 500 mg.

The invention also provides methods for prophylactic prevention of theonset of a bacterial infection, for example an infection caused byStaphylococcus aureus, or by a Neisseria or Clostridium genus bacterium.In a prophylactic method of the invention, a prophylactically effectiveamount of dalbavancin is administered to an individual who may besusceptible to contracting a bacterial infection, for example, through amedical procedure. Often, dalbavancin is administered in an amountsufficient to provide a prophylactically effective plasma level for atleast about 1 day, at least about 3 days, at least about 5 days, or atleast about one week or longer. Dalbavancin compositions may beadministered, for example, parenterally, e.g., via intramuscular (i.m.),intravenous (i.v.), intraperitoneal (i.p.), subcutaneous (s.c.), orintrathecal (i.t.) injection, prior or subsequent to surgery as apreventative step against infection. Dalbavancin compositions may beadministered immediately prior or subsequently to, 1 or more days orabout one week prior or subsequently to, or during an invasive medicalprocedure such as surgery or a stay in a medical care facility such as ahospital to prevent infection. A prophylactic method may be used in anysituation in which it is possible or likely that an individual maycontract a bacterial infection, including situations in which anindividual has been exposed to or is likely to be exposed to abacterially infected individual. For prophylactic methods, dalbavancincompositions may be administered as either a single dose or as two ormore doses of equal or different amount that are administered severaldays to about one week apart. In one embodiment, a dalbavancincomposition may be administered prior to or simultaneously withinsertion of an intravenous catheter in order to prevent a bloodstreamrelated infection.

For prophylactic methods, dalbavancin compositions may be administeredin a single dose or in multiple doses, according to any of the dosingschemes described above. Often, a dalbavancin composition isadministered as a single dose comprising about 0.1 to about 3 grams, orabout 0.1 to about 1 gram, e.g., about 0.25 gram or about 0.5 gram. Inone embodiment, a single dose of about 0.25 gram is administeredintravenously over a time frame of about 2 minutes to about 1 hour,e.g., about 30 minutes. In another embodiment, the dalbavancincomposition is administered intravenously simultaneously withadministration of another pharmaceutical (e.g., antibiotic) treatment.

In any of the therapeutic or prophylactic methods described above, thedalbavancin composition may be administered either simultaneously orsequentially with at least one other antibiotic. In some embodiments, atleast one other antibiotic that is effective (e.g., bactericidal)against one or more Gram-negative bacterial species and/or aGram-positive bacterial strain against which dalbavancin is noteffective is administered in addition to dalbavancin. In someembodiments, dalbavancin and at least one antibiotic that is effective(e.g., bactericidal) against at least one Gram-negative bacterialspecies is administered as a mixture in the dalbavancin composition.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions formulated foradministration of dalbavancin according to the methods described above.Pharmaceutical compositions of the invention may be in the form of aunit dose of dalbavancin that includes an amount of dalbavancinsufficient to provide a therapeutically or prophylactically effectiveplasma level of dalbavancin for several days, often at least about 3days, at least about 5 days, or at least about one week or longer whenthe composition is administered to an individual, and a pharmaceuticallyacceptable carrier. Generally, a therapeutically or prophylacticallyeffective plasma level of dalbavancin is at least about 4 mg per literof plasma. Plasma levels of dalbavancin may be measured by well knownmethods in the art, such as those described above.

Dalbavancin may optionally be in a pharmaceutically acceptable form foradministration to an individual, optionally as a pharmaceuticallyacceptable, non-toxic salt.

Examples of suitable salts of dalbavancin include salts formed bystandard reaction with both organic and inorganic acids such as, forexample, hydrochloric, hydrobromic, sulfuric, phosphoric, acetic,trifluoroacetic, trichloroacetic, succinic, citric, ascorbic, lactic,maleic, glutamic, camphoric, glutaric, glycolic, phthalic, tartaric,lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic,picric, benzoic, cinnamic, and the like acids. Representative examplesof bases that can form salts with dalbavancin include alkali metal oralkaline earth metal hydroxides such as sodium, potassium, calcium,magnesium, and barium hydroxide, ammonia and aliphatic, alicyclic, oraromatic organic amines such as methylamine, dimethylamine,diethylamine, ethanolamine, and picoline. (See, for example, U.S. Pat.No. 5,606,036.)

In some embodiments, a pharmaceutically acceptable aqueous formulationof dalbavancin is provided that is suitable for parenteraladministration, such as, for example, intravenous injection. Forpreparing such an aqueous formulation, methods well known in the art maybe used, and any pharmaceutically acceptable carriers, diluents,excipients, or other additives normally used in the art may be used. Inone embodiment, a pharmaceutically acceptable aqueous formulation forintravenous injection includes 5% dextrose.

A pharmaceutical composition for parenteral administration includesdalbavancin and a physiologically acceptable diluent such as deionizedwater, physiological saline, 5% dextrose, water miscible solvent (e.g.,ethyl alcohol, polyethylene glycol, propylene glycol, etc.), non-aqueousvehicle (e.g., oil such as corn oil, cottonseed oil, peanut oil, andsesame oil), or other commonly used diluent. The formulation mayadditionally include a solubilizing agent such as polyethylene glycol,polypropylene glycol, or other known solubilizing agent, buffers forstabilizing the solution (e.g., citrates, acetates, and phosphates)and/or antioxidants (e.g., ascorbic acid or sodium bisulfite). (See, forexample, U.S. Pat. No. 6,143,739.) Other suitable pharmaceuticalcarriers and their formulations are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. As is known in the art,pharmaceutical preparations of the invention may also be prepared tocontain acceptable levels of particulates (e.g., particle-free) and tobe non-pyrogenic (e.g., meeting the requirements of an injectable in theU.S. Pharmacopeia).

In one embodiment, a pharmaceutical composition is provided bydissolving a dried (e.g., lyophilized) dose of dalbavancin, oftencontaining a stabilizer or mixture of stabilizers, in an amount of waterand preferably deionized water in a volume sufficient forsolubilization. Typically, the amount of water sufficient forsolubilization is approximately 10 mL and the resulting pH of thedalbavancin solution is above 3.0, and about 3.5 to 4.5. Diluting thissolution by adding it to a second amount of an aqueous diluent, oftencontaining 5% dextrose, such as an amount contained in a drip bag forintravenous administration, raises the pH of the dalbavacin solution toabout 5 to 5.5. In another embodiment, the pH of the dalbavancinsolution in a drip bag is about 4.5. The second amount of aqueoussolution may be deionized or sterile, or both deionized and sterile. Inone embodiment, the aqueous diluent is 5% dextrose.

Pharmaceutical compositions for parenteral administration may be made upin sterile vials containing one or more unit doses of dalbavancin in atherapeutically or prophylactically effective amount as described above,optionally including an excipient, under conditions in whichbactericidal effectiveness of dalbavancin is retained. The compositionmay be in the form of a dry (e.g., lyophilized) powder. Prior to use, aphysiologically acceptable diluent may be added and the solutionwithdrawn via syringe for administration to a patient. A pharmaceuticalformulation as described above may be sterilized by any acceptable meansincluding, for example, e-beam or gamma sterilization methods, or bysterile filtration.

A typical formulation for parenteral administration may includedalbavancin at a concentration such as about 0.1 to about 100 mg, about0.5 to about 50 mg, about 1 to about 10 mg, or about 2 to about 4 mg ofdalbavancin per ml of final preparation.

In some embodiments, a pharmaceutical composition in accordance with theinvention includes a mixture of dalbavancin and one or more additionalantibiotics. Preferably, at least one non-dalbavancin antibiotic in themixture is effective (e.g., bactericidal) against one or more species ofGram-negative bacteria, such as, for example, azthreonam, and/or againstone or more Gram-positive bacterial strains against which dalbavancin isnot effective, such as, for example, ilnezolide or daptomycin. Themixture may also include a pharmaceutically acceptable carrier asdescribed above.

In some embodiments, pharmaceutical compositions of the inventioninclude one or more stabilizing substances which inhibit degradation ofone or more of the components of dalbavancin to less active or inactivematerials, for example, MAG. As used herein, “stabilizing substance” or“stabilizer” refers to a substance that stabilizes the level of one ormore of the constituent components of dalbavancin, for example, B₀, inthe composition. A “stabilizing effective amount” refers to an amount ofa stabilizer sufficient to enhance long-term stability of one or morecomponents of a dalbavancin composition. In some embodiments, astabilizing effective amount may be provided by a mixture of two or morestabilizing substances, each of which alone is not present in an amountsufficient to provide a stabilizing effect.

Examples of stabilizers include, for example, nonionic substances suchas sugars, e.g., mono-, di-, or polysaccharides, or derivatives thereof,sugar alcohols, or polyols. Such stabilizing substances include, forexample, mannitol, lactose, sucrose, sorbitol, glycerol, cellulose,trehalose, maltose, raffinose, or mixtures thereof.

In one embodiment, the pharmaceutical composition includes a weightratio of 1:2 mannitol:dalbavancin. In another embodiment, thepharmaceutical composition includes a weight ratio of 1:1:4mannitol:lactose:dalbavancin. Surprisingly, it has been found that acombination of mannitol and lactose provides a greater stabilizingeffect than either substance alone. Often, the pH of a pharmaceuticalcomposition of the invention is, for example, about 3 to about 5, forexample about 3.5 or about 4.5.

In some embodiments, one or more procedures may be employed to reduceformation of MAG. For example, freeze drying of dalbavancin in thepresence of a stabilizing substance, such as mannitol, may be employedto reduce the amount of MAG formed.

Storage of dalbavancin compositions is often at lower than ambienttemperature, such as at about 5° C., to enhance stability.

Improved Efficacy and Reduced Side Effects

Weekly dosing of dalbavancin at high dosage levels (i.e., resulting insurprising high and long-lasting serum levels) shows a surprisingly goodsafety profile, similar to, or better than, that observed with thestandard therapy of lower doses of conventional antibiotics administereddaily or even 2-4 times daily, as demonstrated by the Examples herein. Asurprisingly high dosage (i.e., resulting in surprising high andlong-lasting serum levels) of dalbavancin may be administered, with lessfrequency than other antibiotics, and without adverse side effects,enabling improved efficacy and patient compliance.

As discussed in Example 1, treatment with dalbavancin results in a lowincidence of adverse events. Serious adverse events include any adversedrug experience occurring at any dose that results in death, islife-threatening, results in hospitalization or prolongation of existinghospitalization, or persistent or significant disability or incapacity.In the Phase II trial described in Example 1, 90% of adverse reactions,such as diarrhea, nausea, hyperglycemia, limb pain, vomiting, andconstipation, were mild to moderate in severity. Use of dalbavancin inthe trial in Example 1 resulted in no serious adverse events related tostudy drug treatment.

Kits

The invention also provides kits for use in methods of treatment orprophylaxis of bacterial infections. The kits include a pharmaceuticalcomposition of the invention, for example including at least one unitdose of dalbavancin, and instructions providing information to a healthcare provider regarding usage for treating or preventing a bacterialinfection. Instructions may be provided in printed form or in the formof an electronic medium such as a floppy disc, CD, or DVD, or in theform of a website address where such instructions may be obtained.Often, a unit dose of dalbavancin includes a dosage such that whenadministered to an individual, a therapeutically or prophylacticallyeffective plasma level of dalbavancin is maintained in the individualfor at least 5 days. In some embodiments, a kit includes two unitdosages to be administered at least 5 days apart, often about one weekapart, often including a first dosage of dalbavancin that is about 1.5to about 3 times higher than the second dosage. Dalbavancin is oftenincluded as a sterile aqueous pharmaceutical composition or dry powder(e.g., lyophilized) composition.

Suitable packaging is provided. As used herein, “packaging” refers to asolid matrix or material customarily used in a system and capable ofholding within fixed limits a dalbavancin composition suitable foradministration to an individual. Such materials include glass andplastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles,vials, paper, plastic, and plastic-foil laminated envelopes and thelike. If e-beam sterilization techniques are employed, the packagingshould have sufficiently low density to permit sterilization of thecontents.

Kits may also optionally include equipment for administration ofdalbavancin, such as, for example, syringes or equipment for intravenousadministration, and/or a sterile solution, e.g., a diluent such as 5%dextrose, for preparing a dry powder (e.g., lyophilized) composition foradministration.

Kits of the invention may include, in addition to dalbavancin, anon-dalbavancin antibiotic or mixture of non-dalbavancin antibiotics,for use with dalbavancin as described in the methods above.

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning.

-   -   AcOH=acetic acid    -   AcONa=sodium acetate    -   aq.=aqueous    -   AST=aspartate amino transferase    -   ALT=alanine amino transferase    -   BV=bed volume    -   Cv=coefficient of variation    -   d=diameter    -   D=dalton    -   DCC=dicyclohexylcarbodiammide    -   DMEPA=3-(dimethylamino)-propylamine    -   DMSO=dimethyl sulfonamide    -   eq=equivalents    -   EU=endotoxin units    -   G=gram    -   GC=gas chromatography    -   HCl=hydrochloric acid    -   H₂O=water    -   HOBT=1-hydroxybenzothiazole hydrate    -   HPLC=high performance liquid chromatography    -   H₂SO₄=sulfuric acid    -   IPA=isopropylamine    -   IU=international unit    -   KF=potassium fluoride    -   Kg=kilogram    -   L=liter    -   LC/MS/MS=liquid chromatography/mass spec/mass spec    -   LDH=lactate dehydrogenase    -   LSC=liquid scintillation counting    -   m³=cubic meter    -   MeOH=methanol    -   Mg=milligram    -   mL=milliliter    -   mol=molar    -   MW=molecular weight    -   N=normal    -   NaOH=sodium hydroxide    -   NMP=N-methyl-2-pyrrolidone    -   QTD=quantitative tissue distribution    -   Rt=retention time    -   Sd=standard deviation    -   TEA=triethylamine

The following examples are intended to illustrate but not limit theinvention.

EXAMPLES Example 1 Efficacy and Safety of Once Weekly Dalbavancin inDeep Skin and Soft Tissue Infections

This randomized, controlled study evaluated the safety and efficacy oftwo dose regimens of dalbavancin. Adult patients with skin and softtissue infections (SSTI) involving deep skin structures or requiringsurgical intervention were randomized to three groups: Study arm 1received 1100 mg of dalbavancin via intravenous injection (IV) on day 1;Study arm 2 received 1 g of dalbavancin IV on day 1 and 500 mg ofdalbavancin IV on day 8; Study arm 3 received “standard of care.”Clinical and microbiological response and adverse events were assessed.

Populations for Analysis

There were 62 patients randomized into the study; all received at leastone dose of study medication. Four study populations were evaluated forsafety and efficacy and were defined as follows: The intent-to-treat(ITT) population included all patients who received at least one dose ofstudy drug (all randomized study subjects). The microbiologicalintent-to-treat (MITT) population were all ITT patients who had aculture-confirmed Gram-positive pathogen at baseline. Theclinically-evaluable population were defined as those who 1) fulfilledall study entry criteria, 2) had no change in antimicrobial therapy forGram-positive infection following Day 4, except for oral step-downtherapy (only applied to standard of care group), 3) returned for thefollow-up (FU) assessment visit (unless a treatment failure), and 4) didnot receive a non-protocol approved concomitant antimicrobial (unless atreatment failure). The microbiologically-evaluable population was thesubset of clinically-evaluable patients who had a culture-confirmedGram-positive pathogen at baseline.

The study populations are shown in Table 2.

TABLE 2 Study Populations for Dalbavancin SSTI Treatment Study arm 2Study arm 1 Dalbavancin Dalbavancin 1000 mg day 1, Study arm 3Populations 1100 mg day 1 500 mg day 8 “Standard of care” Randomized 2021 21 ITT Treated   20 (100%)   21 (100%)   21 (100%) Completed 18/20(90%) 20/21 (95.2%) 21/21 (100%) Study Clinically eval 16/20 (80%) 17/21(81%) 21/21 (100%) at EOT Clinically eval 13/20 (65%) 17/21 (81%) 21/21(100%) at FU MITT 14/20 (70%) 13/21 (61.9%) 14/21 (66.7%) Micro eval at13/20 (65%) 11/21 (52.4%) 14/21 (66.7%) EOT Micro eval at 11/20 (55%011/21 (52.4%) 14/21 (66.7%) FU ITT—intent-to-treat MITT - subset of ITTpopulation with culture confirmed Gram-positive infection EOT—end oftreatment FU—follow up

The median age of the subjects was 50-55 years (range 18-86 years).There were no apparent differences in age across the treatment arms.There were differences in gender across treatment arms, but overall thestudy enrolled equal numbers of men and women. The patient populationwas predominantly Caucasian. These results were consistent for both theITT and clinically evaluable populations.

62 patients were enrolled, 20 in Study arm 1 and 21 each in Study arms 2and 3. The most common comparators for standard of care wereclindamycin, ceftriaxone, vancomycin and cefazolin. Mean duration oftreatment in Study arm 3 was 15 days.

Baseline Pathogens and Susceptibility

Of the 62 ITT patients, 66% (14 single-dose dalbavancin, 13 two-dosedalbavancin, 14 standard of care) had a pretherapy Gram-positivepathogen isolated (MITT population). The most common pathogen was S.aureus. The distribution of pathogens at baseline is shown in Table 3.

TABLE 3 Baseline Gram-positive Pathogens and Dalbavancin MIC Range forthe MITT Population Single dose Two-dose Number with DalbavancinDalbavancin Standard of Care Pathogen (1100 mg) (1000/500 mg) Regimens(MIC) (N = 14) (N = 13) (N =14) All S. aureus 13 11 10 (0.12) (0.12)(0.016-0.25) Methicillin- 7 6 8 sensitive Methicillin- 6 5 2 resistantGroup B 0 2 2 streptococcus (0.016) (0.016) Streptococcus 0 1 1 pyogenes(0.016) (0.016) Miscellaneous 3 2 4 Streptococcus (0.016) (0.016)(0.016) and non- typeable strains

Clinical and Microbiological Responses

The effectiveness of the three treatment regimens was determined byassessing the patients' clinical response and the documented or presumedmicrobiological responses. The primary efficacy endpoint was clinicalresponse at the follow-up visit for the clinically evaluable population.Clinical response, for both EOT and FU visits, was categorized assuccess (cure or improvement) or failure (including indeterminateresults). Patients classified as successes must not have receivedadditional systemic antibacterial treatment for their infection. Failurewas defined as persistence of one or more local or systemic signs andsymptoms of SSTI such that treatment with new or additional systemicantibacterial agents was required for the SSTI.

Microbiological outcome, a secondary efficacy variable, was assessed inthe subpopulation of patients who had microbiologically documented SSTI(i.e., at least one identified baseline pathogen). A microbiologicresponse was assessed for each Gram-positive pathogen identified atbaseline (i.e., eradication, presumed eradication, persistence, presumedpersistence). For patients for whom follow-up cultures were notperformed, the microbiologic responses for baseline pathogens werepresumed on the basis of the clinical response. Microbiologic responseby patient at the EOT and FU visits was graded as success (i.e., allGram-positive organisms eradicated or presumed eradicated) or failure(i.e., at least one Gram-positive organism persisted or presumed to havepersisted, multiple pathogens with partial eradication). At both the EOTand FU visits, colonization and superinfection were assessed. At the FUvisit, a patient's bacteriological response could also includerecurrence.

Clinical Efficacy

Clinical success rates are shown in Table 4. In the clinically-evaluablepopulation, 61.5% of patients in the single-dose dalbavancin, 94.1% inthe two-dose dalbavancin, and 76.2% in the standard of care group wereclassified as successes at the time of the FU assessment. In anexploratory subanalysis of those patients categorized with deep orcomplicated SSTI at baseline, two-dose dalbavancin therapy also provideda higher clinical success rate (93.8%), compared with the single-dosedalbavancin and standard of care therapies, 58.3% and 73.7%,respectively.

Similar success rates at both the EOT and FU assessments were found inthe supportive ITT and microbiologically-evaluable populations with aconsistent trend towards a more favorable response following treatmentwith two-dose dalbavancin (Table 3). For the MITT population, clinicalsuccess rates at the FU assessment for those with methicillin-resistantS. aureus (MRSA) were 50% ( 3/6) for single-dose dalbavancin, 80% (⅘)for two-dose dalbavancin, and 50% (½) for patients treated with astandard of care regimen

TABLE 4 Clinical Success Rates by Analysis Population and TreatmentGroup Single-dose (1100 mg) Two-dose (1000/500 mg) Standard of CarePopulation Dalbavancin Dalbavancin Regimens ITT at EOT 15/20 (75.0)19/21 (90.5) 17/21 (81.0) ITT a FU 12/20 (60.0) 19/21 (90.5) 16/21(76.2) MITT at EOT 10/14 (71.4) 12/13 (92.3) 10/14 (71.4) MITT at FU 7/14 (50.0) 12/13 (92.3)  9/14 (64.3) Clinically evaluable at 13/16(61.5) 16/17 (94.1) 17/21 (81.0) EOT Clinically evauable at  8/13 (61.5)16/17 (94.1) 16/21 (76.2) FU Microbiologically 10/13 (76.9) 10/11 (90.9)10/14 (71.4) evaluable at EOT Microbiologically  6/11 (54.5) 10/11(90.9)  9/14 (64.3) evaluable at FU

Microbiologic Efficacy

The success rates of the different treatment regimes with respect todifferent pathogens is shown in Table 5. For themicrobiologically-evaluable population, eradication/presumed eradicationrates at the FU assessment for all organisms were 58.3% ( 7/12) forsingle-dose dalbavancin, 92.3% ( 12/13) for two-dose dalbavancin, and70.6% ( 12/17) for patients in the standard of care group. For isolatesthat persisted, there was no change in dalbavancin MIC. At FU, S. aureuseradication rates were higher for the two-dose dalbavancin group (90%)compared with single-dose dalbavancin (50%) and standard of care (60%)treatments. Similar findings were observed for the MITT population;two-dose dalbavancin eradicated 80% of MRSA isolates (Table 5).

TABLE 5 Success Rates by Pathogen for Microbiologically ITT Populationat FU Assessment Single-dose (1100 mg) Two-dose (1000/500 mg) Standardof Care Dalbavancin Dalbavancin Regiments Total organisms  7/16 (43.8%)14/16 (87.5%)  12/17 (70.6%)  All. S. aureus 5/13 (38%)  9/11 (82%) 6/10 (60%)  Methicillin- 2/7 (29%) 5/6 (83%) 5/8 (63%) sensitiveMethicillin-resistant 3/6 (50%) 4/5 (80%) 1/2 (50%) Miscellaneous 2/3(67%)  4/4 (100%) 5/7 (71%) streptocococcus species

For the microbiologically-evaluable and MITT populations, themicrobiological success rates at EOT and FU are summarized in Table 6.Comparable microbiologic success rates were reported at both visits forpatients treated with two-dose dalbavancin and standard of care regimens(approximately 64% to 77%), whereas those given a single dose ofdalbavancin had lower rates of success (<40%). The microbiologic successrates at EOT/FU in the microbiologically-evaluable population paralleledclinical response findings: 38.5%/27.3% for single-dose dalbavancin,72.7%/72.7% for two-dose dalbavancin, and 71.4%/64.3% for standard ofcare therapy. Similar findings were observed for the MITT population(data not shown).

TABLE 6 Microbiologic Success Rates Single-dose (1100 mg) Two-dose(1000/500 mg) Standard of Care Dalbavancin Dalbavancin Regimens MITTpopulation EOT 5/14 (35.7) 10/13 (76.9)  10/14 (71.4) FU 3/14 (21.4)9/13 (69.2)  9/14 (64.3) Microbiologically evaluable population EOT 5/13(38.5) 8/11 (72.7) 10/14 (71.4) FU 3/11 (27.3) 8/11 (72.7)  9/14 (64.3)

Pharmocokinetic Analysis

For patients randomized to the dalbavancin treatment groups, 5 ml ofblood was obtained on Day 8 for determination of dalbavancin plasmaconcentrations. For patients randomized to receive a 500 mg dalbavancindose on Day 8, blood was obtained just prior to administration of thesecond dose. Additional 5 ml blood samples were obtained on Days 10 and24 for patients were randomized to the single-dose dalbavancin group andon Days 20 and 34 for those who received two doses of dalbavancin.

Dalbavancin plasma concentrations were determined using validated liquidchromatography and mass spectrophotometer methods. The lower limit ofquantitation was 500 ng/ml for plasma.

Mean dalbavancin concentrations collected on Study days 8, 10, and 24 inthe single-dose regimen were 31.1±7.1, 25.2±4.8, and 10.2±3.5 mg/l(mean±SD), respectively. Dalbavancin concentrations following thetwo-dose regimen on Study days 8 (prior to the second dose), 20, and 34were 30.4±8.2, 21.2±10.0, and 9.0±4.4 mg/l, respectively. As expected,all patients had dalbavancin concentrations of greater than 20 mg/lthrough the first week following the first dose, and levels above 20mg/l were maintained for an additional week with an additional dose of500 mg IV on day 8. Generally, minimum bactericidal concentrations areabout 4 to 10 mg/l.

Safety Evaluation

Each patient who received at least one dose of study drug (ITTpopulation) was evaluated for drug safety through monitoring of adverseevents (AE), including abnormal clinical laboratory test results andvital signs. AE were rated by the investigator as to their severity(mild, moderate, severe, life-threatening), and by the relationship tothe study drug (not related, unlikely related, possibly related, orprobably related).

A summary of the AE data is presented in Table 7. The majority ofadverse reactions (90%) were considered mild to moderate in severity.All serious adverse reactions (8 events in 5 patients) were unrelated tostudy drug treatment. Approximately 59% of all patients who reported atleast one treatment-emergent AE (19 single-does dalbavancin, 16 two-dosedalbavancin, 21 standard of care) experienced an event that wascategorized by the investigator as possibly or probably related to studydrug. Specifically, drug-related AEs were reported in 11 (55%)single-dose dalbavancin, 10 (48%) two-dalbavancin, and 12 (57%) standardof care patients. The most frequently reported drug-related AE in boththe dalbavancin and standard of care treatment groups were diarrhea andnausea. A summary of types of AEs observed for the different treatmentgroups is presented in Table 8.

No dalbavancin-treated patient discontinued treatment prematurely due toan AE. Three of 21 (14%) patients on a standard of care regimentdiscontinued treatment prematurely due to an AE, including one patientwho developed urticaria on Day 1 which was probably drug related and twopatients who had AE unrelated to study drug (superinfection with P.aeruginosa and elevated vancomycin trough level).

TABLE 7 Summary of Adverse Event (AE) Data Single-dose Two-dosedalbavancin dalbavancin Standard of Care (N = 20) (N = 21) (N = 21) ≧1AE 95% 76.2% 100% % AE severe 15%  9.5%  4.8% ≧ AE 55%   48%  57%possibly/probably related to treatment AE leading to 0 0 14.3% discontinuation of study medication ≧ severe AE 2 (10%) 2 (9.5%) 1(4.8%) 

TABLE 8 Most Common Adverse Events Single-dose Two-dose dalbavancindalbavancin Standard of Care (N = 20) (N = 21) (N = 21) Diarrhea 20%9.5% 28.6%  Nausea 10% 28.6% 9.5% Hyperglycemia 5% 14.3%  19% Limb Pain10% 9.5% 9.5% Vomiting 10% 14.3% 4.8% Constipation 5% 4.8% 14.3% 

Discussion

This open-label, randomized Phase II trial shows that dalbavancin iseffective for the treatment of adults with SSTI. The majority ofenrolled patients had deep or complicated infections (>90%) andinfections that required surgical intervention (˜70%), whileapproximately 45% had underlying diabetes mellitus.

Two weekly doses of dalbavancin had a numerically higher clinicalresponse rate than either a single-dose of dalbavancin or the standardof care regimen. Data from both ITT and clinically-evaluable populationssuggests that a regimen of two sequential weekly injectable doses ofdalbavancin (1000 mg, 500 mg weekly) is effective in the treatment ofSSTIs. The standard of care group was treated for a median duration of13 days. At follow-up, 94% of clinically-evaluable patients treated withtwo-dose dalbavancin were considered clinical successes versus 76% ofthose given a standard of care regimen and 61.5% of patients receivingsingle-dose dalbavancin.

S. aureus was the most frequently isolated organism at baseline. In thistrial, approximately 83% of patients were infected with S. aureus and38% of all S. aureus strains were MRSA. Most infections (80%) werecaused by a single pathogen. The MICs for dalbavancin againstGram-positive isolates, including MRSA, ranged from 0.016 to 0.25 mg/L.

Microbiological success rates paralleled those of clinical response forthe clinically-evaluable population. For all organisms combined,treatment with the two-dose weekly dalbavancin regimen provided highereradication rates at the 2 week post-therapy assessment (92%) comparedwith single-dose dalbavancin (58%) and standard of care therapies (71%).Overall, rates of S. aureus eradication were observed in 90%, 50%, and60% of patients, respectively. For the MITT population, rates oferadication for MRSA were 80% for the two-dose dalbavancin regimenversus 50% for both the single-dose dalbavancin and standard of caretherapies.

Concentrations of dalbavancin obtained at the end of the single-dose andtwo-dose weekly treatment periods (Day 10 or Day 20, respectively) weresimilar suggesting little drug accumulation following the second weeklydose. The higher rate of clinical success observed with the two-doseregimen is suggestive of time-dependent killing wherein sustained levelsof drug or drug exposure were provided with two doses of dalbavancinseparated by one week. Dalbavancin plasma levels measured at the end ofthe weekly dosing interval were substantially greater than the reportedMIC₉₀ for pathogens responsible for the majority of SSTIs (<0.03 to 0.5mg/L), including those found in this trial. These levels were also abovethe minimum bactericidal concentrations of 4 to 10 mg/l.

The overall rate of adverse reactions was similar for both dalbavancinregimens and the standard of care group. Gastrointestinal drug-relatedadverse events (i.e., diarrhea and nausea) were most commonly reportedacross the three treatment groups. The majority of these events weremild and self-limited. No dalbavancin-treated patient was withdrawn fromthe study early due to an adverse reaction, nor were any serious adverseevents attributable to the glycopeptide reported, yet 14% of thestandard of care group withdrew due to adverse effects. The novel dosageregimen thus had reduced adverse side effects in comparison to thestandard of care. The data from this trial found no evidence thatdalbavancin induces any degree of clinically significant hepatotoxicityor nephrotoxicity.

The two-dose dalbavancin regimen appears effective for treatment ofpatients with complicated SSTIs. Dalbavancin at both doses was welltolerated in this clinical trial, with an adverse event profile similarto that of the standard of care group.

Example 2 Pharmacokinetics and Renal Excretion of Dalbavancin in HealthySubjects

The primary objectives of this study were to characterize thepharmacokinetics of dalbavancin and to calculate the extent of renalexcretion in healthy subjects receiving a therapeutic dose of the drug.This was an open label, non-comparative, study.

Study Drug Treatment

Healthy male or female subjects between 18 and 65 years of age wereadministered a single 1000 mg IV dose of dalbavancin infused over 30minutes.

Six subjects, one female and five male, were enrolled, received studymedication, and completed all aspects of the study. Three subjects wereCaucasian and three subjects were African-American. Mean age was 29.8years (range 22 to 63). Mean height was 68.6 inches (range 63 to 75) andmean weight was 179.6 lbs (140 to 244).

Pharmacokinetics

Blood and urine (24-hr collections) were collected on study days 1, 2,3, 4, 5, 6, 7, 14, 21, 28, and 42. Blood samples were drawn intoheparinized tubes and centrifuged. Plasma was separated and storedfrozen at −20° C. until time of assay. Plasma and urine samples wereassayed for dalbavancin using validated LC/MS/MS methods. The lowerlimit of quantitation of the assay was 500 ng/mL for urine and plasma.

Dalbavancin pharmacokinetic parameters were estimated bynon-compartmental methods using the WinNonlin™ software (PharsightCorporation). The peak concentration (C_(max)) values were obtaineddirectly from the observed data. The area under the plasmaconcentration-time curve (AUC) was calculated using the lineartrapezoidal rule. Clearance (CL) was computed as dose/AUC. Theelimination half life (t_(1/2)) was estimated by linear regression ofthe log-linear portion of the log concentration versus time curve.Estimates of the volume of distribution (Vz) was calculated using theregression parameters, while the volume of distribution at steady state(Vss) was calculated from the area under the first moment curve (AUMC)multiplied by the dose and divided by AUC. The cumulative amount ofdalbavancin excreted in urine was determined as the integrand of theurine excretion rate (AURC). CL_(R), or renal clearance, was calculatedas the ratio:

CL _(R)=AURC/AUC.

Plasma concentrations of dalbavancin versus time are shown for allsubjects in FIG. 1. Pharmacokinetic parameters are presented in Table 9.Concentrations were similar across all subjects. Peak plasmaconcentrations were approximately 300 mg/L and were achieved immediatelyfollowing the end of infusion. Dalbavancin shows an apparent volume ofdistribution of more than 10 L and is assumed to be well distributed inthe extracellular fluid.

Dalbavancin was slowly eliminated with a t_(1/2) of 9-12 days. The totaldrug clearance was 0.0431±0.0074 L/hr. The estimated fraction of drugexcreted unchanged into urine was 42% of the administered dose, andrenal clearance was estimated as 0.018 L/h. The variability observedacross subjects was low with a coefficient of variation of less than 22%across all pharmacokinetic parameters.

TABLE 9 Pharmacokinetic parameters Cmax T_(1/2) AUC V_(z) CL V_(SS) AURC% Renal CL_(R) Mg/L h mg · h/L L L/h L mg Excretion L/h Mean 301 25723843 16.0 0.0431 11.5 419 41.9 0.0181 Sd 65 21 4526 3.1 0.0074 2.13 272.7 0.0036 CV % 21.6 8.1 19.0 19.5 17.1 18.6 6.4 6.4 20.1 Min 243 22719844 11.7 0.0332 8.58 379 37.9 0.0130 Max 394 282 30100 19.6 0.050413.9 448 44.8 0.0226

Safety Assessments

Adverse events were recorded and assessed for severity and relationshipto study drug. Laboratory data (chemistry panel, CBC with differential,urinalysis) were collected and assessed for changes from baseline andout-of-range values. ECG, physical examination, and vital signs wereobtained, and changes from baseline were assessed.

Dalbavancin was well-tolerated in this study. No subject deaths orserious adverse events were reported during this study and no subjectwas prematurely withdrawn from study due to an AE.

All volunteers reported at least one AE, all of mild intensity. Threevolunteers reported AEs that were possibly related to study medication:elevated ALT (value 46 IU/L, upper limit of normal 40 IU/L) in onesubject; eosinophilia (value 0.5×10³/μL, upper limit of normal0.4×10³/μL), elevated LDH (value 303 IU/L, upper limit of normal 90IU/L), elevated ALT (value 54 IU/L, upper limit of normally 40 IU/L),elevated AST (value 42 IU/L, upper limit of normal 40 IU/L) all in onesubject; and tinnitus in one subject.

No trends were seen for post-baseline hematology, chemistry, vitalsigns, and ECG results.

Discussion

A single 1000 mg IV dose of dalbavancin was well-tolerated. Following asingle intravenous infusion of 1000 mg, plasma concentrations ofdalbavancin above 45 mg/l are maintained for at least seven days. Thisis above concentrations known to be bactericidal (4-32 mg/l). Thissupports the use of dalbavancin as a once-weekly regimen. The urinaryelimination profile indicates that renal excretion is an importantelimination pathway, with approximately 40% excreted in urine. Thisfinding is consistent with observations in animals. Since the kidneysare not the exclusive elimination route, a dosing adjustment fordalbavancin may not be necessary in renally impaired patients.

Example 3 Protein Binding of Dalbavancin Using Isothermal TitrationMicrocalorimetry

Binding of dalbavancin to proteins was measured by isothermal titrationmicrocalorimetry (ITC) in 20 mM phosphate, 150 mM NaCl, pH 7.4 at 25 and37° C. using a Microcal VP-ITC instrument. In a typical experiment,25×10 μl of protein (˜150 μM) was injected into a calorimeter cellcontaining dalbavancin solution (˜5 μM). Actual protein and dalbavancinconcentrations were determined by measuring absorbence at 280 nm.Control experiments included injections of protein into buffer (in theabsence of dalbavancin) to account for the heats of dilution of proteinunder identical conditions. For comparison, similar experiments withsome necessary modifications were performed using teicoplanin.

Experiments with dalbavancin were conducted with each of the followingproteins: human albumin; dog albumin; rat albumin; bovine albumin; andhuman a-glycoprotein. Teicoplanin was studied with human albumin anda-glycoprotein. A comparison of binding affinities at two differenttemperatures is shown in Table 10.

TABLE 10 Comparison of apparent binding affinities (Ka, × 10⁵ M⁻¹⁾ 25°C. 37° C. Dalbavancin Human albumin 1.35 (± 0.2) 1.33 (± 0.15) Ratalbumin  3.1 (± 0.5)  2.8 (± 1.8) Dog albumin 0.62 (± 0.09) 0.50 (±0.13) Bovine albumin 1.38 (± 0.14) α-glycoprotein 1.84 (± 0.36)  4.8 (±2.3) Teicoplanin Human albumin 0.96 (± 0.08) α-glycoprotein 0.07 (±0.01) The ± errors quoted are the standard deviations obtained from thefitting routine.

Integrated heat effects, after correction for heats of dilution, wereanalyzed by non-linear regression using a simple single-site bindingmodel with the standard Microcal ORIGIN software package. Raw data(μcal/sec) for each injection were integrated to give the total heateffect per addition, then divided by amount of injectant to givekcal/mole of injectant. The same integration was applied for controldilution effects, and this was subtracted from the actual titrationdata. This provided a differential of the binding curve in which theextent of binding is proportional to the total heat liberated (orabsorbed). This was then analyzed by non-linear regression methods interms of various standard binding models. The simplest model assumessimple non-competitive binding equilibrium, and gives three parameters:

K_(a)(=1/Kdiss) is the binding association (dissociation constant)ΔH=the enthalpy of binding (the size of signal related to binding)N=number of binding sites (assuming the binding model is correct)

Assuming non-competitive binding, N is the (relative) number of moles ofinjectant required to saturate all the available binding sites in thesample. For the dalbavancin experiments, dalbavancin is the “sample” andthe protein (HSA, etc.) is the “injectant.” These preliminary resultsindicate that the binding is relatively weak and, because of the poorsolubility of dalbavancin, it is difficult to determine the bindingstoichiometry (N) unambiguously. However, as seen in FIG. 2, in allcases, the data fits well with N<1 (i.e., less than one to one proteinto dalbavancin). Consequently, a value of N=0.5 means that it only takeshalf as many moles of protein to bind all the dalbavancin than would beexpected. In other words, each protein apparently binds two dalbavancinmolecules. It is possible that dalbavancin forms a dimer that binds 1:1with a protein. Results of binding stoichiometry modeling suggests thattwo dalbavancin molecules are bound to one molecule of protein, unliketeicoplanin, which exhibits 1:1 binding.

Table 11 presents the calculated percent bound for antibioticconcentrations in the range 1-500 μM, assuming physiologicalconcentrations of human serum albumin (6×10⁻⁴ M) and α-glycopeptide(1.5×10⁻⁵ M). To relate this to the clinical situation, the peakconcentration of dalbavancin in man is approximately 300 mg/L, or 165μM.

TABLE 11 Calculated percent Binding of Teicoplanin and Dalbavancin toPlasma Proteins Concentration of antibiotic (μM) Dalbavancin TeicoplaninHuman albumin  1 98.8 98.3  10 98.8 98.3 100 98.7 98.0 165 98.6 ND 25098.5 ND 500 98.0 ND Human α-glycoprotein  1 73.0 9.6  10 68.2 9.1 10026.2 6.0 165 16.9 ND 250 11.4 ND 500 5.9 ND ND = Not done

In these experiments, the binding of dalbavancin to human serum albuminexceeds 98%. The fraction bound is fairly constant across the selectedrange of dalbavancin concentrations, i.e. 1-500 μM. This rangeencompasses the therapeutic concentration in man. Binding of dalbavancinto a-glycoprotein is much greater than that of teicoplanin. Dalbavancindemonstrates high capacity and low affinity for plasma proteins ofdifferent origin, with similar K_(a) values across proteins from allspecies tested. These results help to explain some of the uniquepharmacokinetic characteristics of dalbavancin. The binding andformation of a 2:1 dalbavancin:protein complex also explains theprolonged half-life, and the apparent volume of distribution, whichapproximates extracellular water volume. The low affinity helps explainthe observed in vivo activity, which greatly exceeds what would beexpected for a compound with a free fraction close to 1%. The highcapacity for plasma proteins helps to explain the relatively high plasmaconcentrations achieved in spite of poor solubility of the compound atphysiological pH.

Example 4 Pharmacokinetic Attributes and Tissue Distribution ofDalbavancin in Rats

Two studies were performed in rats administered a single IV infusion of20 mg/kg [³H]-dalbavancin. Excreta and more than 40 different tissueswere collected through 70 days post-administration, and the tissuedistribution and pharmacokinetics of drug-derived radioactivity weredetermined.

HPLC-purified [³H]-dalbavancin was used for these studies. Radiolabeleddrug was produced via tritium exchange and purified by HPLC.

Rat Mass Balance Study

Mass balance studies were conducted to determine the excretion patternof dalbavancin following a single intravenous (IV) infusion ofdalbavancin in male rats.

Fifteen male Sprague-Dawley rats received a single IV dose of³H-dalbavancin (20 mg/kg, 100 μCi/rat). Following dose administration,urine and feces were collected at 24 hour intervals to 14, 36, and 70days after the dose (3 rats/final collection time). Water and methanolcage washes were also collected. Carcasses were analyzed at the end ofthe collection period. All samples were analyzed for total radioactivitycontent by liquid scintillation counting (LSC).

Following IV administration of ³H-dalbavancin in rats, drug-derivedradioactivity was eliminated in both urine (˜⅔ of excretedradioactivity) and feces (˜⅓ of excreted radioactivity). Approximatelyhalf of the radioactivity administered was eliminated in the urine andfeces within the first week, which is consistent with a plasma t_(1/2)of approximately 1 week. At 70 days post dose, only 4.5% of the doseremained in the carcass. Negligible radioactivity was recovered in thecage washes. Virtually all of the administered radioactivity wasaccounted for (urine, feces, carcass, cage washes, and tritium exchange)during the study.

Rat Quantitative Tissue Distribution (OTD) Study

Quantitative tissue distribution studies were conducted to assess thetissue distribution of dalbavancin following a single IV infusion ofdalbavancin to male rats. Forty-one male Sprague-Dawley rats received asingle IV infusion of ³H-dalbavancin (20 mg/kg, 50 μCi/rat). Rats (3 pertime-point) were euthanized at 12, 24, 48, 72, 96, 120, 144, 168, 336,840, 1176 and 1680 hours post dose for collection of blood, plasma, andtissues (including carcass). All samples were analyzed by LSC.

Concentration-time profiles were determined for more than 40 tissues,including kidney, liver, spleen, blood, plasma, lung, and skin.Concentrations and t_(1/2) values of drug-derived radioactivity intissues, including skin, were comparable to those observed in plasma.Dalbavancin was found to be rapidly and extensively distributed with alltissues having quantifiable concentrations of drug-derived radioactivitywithin 12 hours after post-infusion. Most tissues reached maximumconcentration (C_(max)) within 24 h after the dose. Recoveredradioactivity after 5 days was <5% of the dose in any single tissue. By70 days after the dose, only the carcass retained >1% (2.34%) of theadministered radioactivity. Thus, dalbavancin did not accumulate in anysingle tissue, organ, or blood cellular component. Concentrations ofradioactivity in the CNS were low but detectable in this healthy animalmodel. Dalbavancin was found to penetrate the skin with concentrationsof drug-derived radioactivity that were as high as or higher than inplasma. Blood to plasma ratio of drug-derived radioactivity remainedrelatively constant over time and was <1.

As part of the QTD studies, bile samples were collected from bile ductcannulated rats (4 animals) through 384 h (16 days) post-dose. Almost11% of the dose was recovered in the bile over 384 h after the dose.This represents the majority of the drug-derived radioactivity found infeces.

Example 5 Quantitative Determination of Dalbavancin in Plasma byHPLC-MS/MS

A HPLC-MS/MS method was developed for quantitative measurement ofdalbavancin in plasma, as described below.

Preparation of Dalbavancin Calibration and Quality Control Standards

Stock solutions of dalbavancin were prepared by dissolving dalbavancinin deionized water to prepare a 1000 μg/ml solution, followed by serialdilutions in deionized water to prepare 500, 50 and 10 μg/ml solutions.

Calibration standards of 100, 60, and 40 μg/ml dalbavancin concentrationwere prepared by spiking human plasma with appropriate volumes of a 1000μg/ml dalbavancin stock solution prepared as described above.Calibration standards of 20 and 10 μg/ml concentration were prepared byspiking human plasma with appropriate volumes of a 500 μg/ml dalbavancinstock solution, and a calibration standard of 0.5 μg/ml was prepared byspiking human plasma with an appropriate volume of a 10 μg/ml stocksolution.

Quality control standards of 90 and 30 μg/ml dalbavancin were preparedby spiking human plasma with an appropriate volume of a 1000 μg/mldalbavancin stock solution prepared as described above. A qualitycontrol standard of 1.5 μg/ml was prepared by spiking human plasma withan appropriate volume of a 50 μg/ml solution.

Preparation of Internal Standard Working Solution

A 30 μg/ml working solution of internal standard BI-K0098, which is thediethyl-amino-propyl-amino derivative of A-40926, was prepared asfollows. Approximately 10 mg of BI-K0098 was dissolved in approximately10 ml of mobile phase A (80% of 10 mM Ammonium Formiate/Formic Acid, pH3 (v/v), 10% of Acetonitrile (v/v), and 10% 2-Propanol (v/v)) to make a1000 μg/ml internal standard stock solution. The stock solution (300 μl)was then diluted to a volume of 10 ml with mobile phase A to make a 30μg/ml internal standard solution.

Preparation of Samples for Analysis

Samples were prepared as follows for quantitative determination ofdalbavancin concentration in plasma. To 50 μl of calibration or qualitycontrol standards prepared as described above, 100 μl of internalstandard working standard solution was added and mixed. The mixture waspermitted to equilibrate for five minutes at room temperature, followedby addition of 250 μl of acetonitrile. The mixture was then vortexed for10 seconds, followed by centrifugation for 1 minute at about 10,000 rpmon an ALC micro-centrifugette 4214. Supernatants were transferred toclean tubes and evaporated to dryness in a Savant Speed-Vac System atabout 40° C. Samples were then resuspended in 150 μl of mobile phase A.

Analytical Method

50 μl samples prepared for analysis as described above were injectedinto a Phenomenex Jupiter C₁₈ column (50×2 mm, C₁₈ 5 μm 300 A), andanalyzed under gradient HPLC conditions at a flow rate of 0.3 ml/min.The gradient conditions were: initial, 80% mobile phase A/20% mobilephase B (20% 10 mM Ammonium Formiate/Formic Acid, pH 3 (v/v), 40%Acetonitrile (v/v), 40% 2-propanol (v/v)); 1 minute, 20% mobile phaseA/80% mobile phase B; 2 minutes, 20% mobile phase A/80% mobile phase B;2.5 minutes, back to initial conditions.

The HPLC system was coupled to a PE SCIEX API-2000 triple quadrupolemass spectrometer, with turbo ion spray operating in a positiveionization mode. Air was used to generate a spray in the ion source.Probe temperature was set at 500° C. with nitrogen as curtain gas.Multiple reactions monitoring (MRM) was employed using nitrogen ascollision gas. The analytes were detected by monitoring the followingion transitions: 909.3 Da→1429.3 Da for dalbavancin, and 923.3 Da→1457.3Da for the internal standard (BI-K0098). To avoid mass spectrometercontamination, a post-column flow diversion in the first minute and 2.5minutes after the beginning of the chromatographic run was performed.

Software Sample Control 1.4 was used for the acquisition of dataanalysis and software MacQuan 1.6 was used for the integration ofchromatographic peaks and statistical data evaluation.

Calibration Curves

Linearity of the assay method was assessed by assaying calibrationstandards to generate a calibration curve. The concentration ofdalbavancin in a plasma sample was determined by calculating the peakarea ratio between dalbavancin and the internal standard.

Calibration curves for dalbavancin concentrations over an analyticalrange of 0.5-100 μg dalbavancin/ml of human plasma were constructedusing the equation γ=A+Bχ(weighted 1/x), where A represents intercept ofthe curve, B represents the slope of the curve, χ represents thedalbavancin concentration of calibration standard (μg/ml), and γrepresents the peak area ratio of dalbanvancin to internal standard.Three separate calibration curves were constructed. The results showedthat dalbavancin/internal standard area ratio and dalbavancinconcentrations varied linearly over the analytical range. The lowerlimit of quantitation (LLOQ) was 0.5 μg dalbavancin per ml of humanplasma. The slopes for the calibration curves were reproducible andtheir correlation coefficients were greater than 0.9995.

Stability of Dalbavancin in Plasma

The stability of dalbavancin in plasma samples was tested by analyzingthree replicates quality control standards of human plasma samples,prepared as described above, at two different concentrations, 1.5 and 90μg/ml. Detectable dalbavancin concentration was stable after threecycles of freeze-thaw treatment. Dalbavancin concentration in processedsamples was stable after 24 hours at room temperature. No reduction indalbavancin concentration with respect to time zero samples wasobserved.

Example 6 Dalbavancin Mass Spectroscopy Analysis

The nature of dalbavancin multimers in solution was investigated and theconditions influencing the population ratio of dalbavancin multimer todalbavancin monomer were determined by electrospray ion massspectroscopy (ESI-MS).

Experiments were performed using an Applied Biosystem API III+ massspectrometer equipped with a TurbolonSpray source, a Triple Quadrupoleanalyzer, operating in positive ion mode. The optimized conditions arereported in Table 12 below.

TABLE 12 Instrumental Conditions for Dalbavancin Analysis on AppliedBiosystem API III + Mass Spectrometer. IonSpray Source: IonSpray voltage5000 V Orifice plate 80 V Voltage Curtain gas flow 0.6 L/min Nebulizergas flow 1.2 L/min Liquid flow 5 μL/min Interface heater 60° C. Scanconditions (Q1 scan): Step 0.1 amu Dwell time 1 msec MS analyzer:Interface plate voltage 650 V Q0 road offset voltage 40 V Q1 park mass1000 Q1 resolution 120.8 Q1 delta mass 0.2 Q1 road offset voltage 27 VLens 7 voltage −50 V Q2 road offset voltage −50 V Q3 park mass 1000 Q3resolution 110 Q3 delta mass 0 Q3 road offset voltage −70 V Lens 9voltage −250 V Faraday plate voltage −250 V Channel electron Multipliervoltage −3800 V

Dalbavancin in Solution

Instrumental parameters were tuned on a dalbavancin solution containing0.1 mg/ml of dalbavancin dissolved in a 8:2 water:isopropanol solution.A spectrum of dalbavancin in solution was acquired in the range of500-2000 amu following direct injection of the solution. The resultingspectrum, as seen in FIG. 3, indicates the presence of dalbavancinmultimers. As a non-limiting example, one trace of the spectrum isattributable to a homomultimer of B₀, which is present as a (2 nM+y(⁺3))ion species, where n is a positive integer indicating the multiplicityof the homomultimer, e.g., n=1 when the multimer is a homodimer and n=2when the multimer is a homotetramer, M indicates the mass of themonomer, γ=n and ⁺3 indicates a plus three ion charge. For example, thehomodimer of B₀ is provided when n=1, γ=1, and M=mass of B₀. Thishomodimer species is assigned to a (2M+3) ion trace in the massspectrum.

Influence of Dalbavancin Concentration on the Population Ratio ofDalbavancin Multimer to Monomer

The influence of dalbavancin concentration on the population ratio ofmultimer to monomer was evaluated by mass spectroscopy using theconditions described above. The spectra were acquired by direct infusionof dalbavancin solutions at concentrations of 20, 40, 60, and 80 μg/mL.The intensities of the main peaks were reported as a function ofdalbavancin concentration and the population ratios of dalbavancinmultimer to monomer were determined, as shown in FIG. 4.

The data indicate that the population ratio of dalbavancin multimer todalbavancin monomer increases with increasing concentration. This mayhelp to explain the high drug loading capacities that may beadministered to an individual. The role of multimer as a depot ofmonomer may decrease the tendency of higher concentration samples toform precipitates and enhance the concentrations which may beadministered to an individual. The presence of multimers may also allowrapid administration of a dose of dalbavancin to an individual.

A non-limiting example of a method of determining the population ratioof dalbavancin multimer to monomer is provided, for example, bydetermining the ratio between peak intensities of ions A and B as shownin FIG. 3. Dividing the intensity of peak A by the intensity of peak Bprovides one measure of the population ratio of dalbavancin multimer tomonomer.

Influence of pH on the Population Ratio of Dalbavancin Multimer toMonomer

The influence of solution pH on the population ratio of dalbavancinmultimer to monomer was evaluated at the instrumental conditionsdescribed above and at the following solution pH values: 2.5, 3.0, 3.5,4.0, 4.5, 5.0, and 5.5. The population ratio of dalbavancin multimer tomonomer was determined at each of the pH values and plotted against pH,as seen in FIG. 5. It was determined that the population ratio ofdalbavancin multimer to monomer increases with increasing pH.

While not to be limited to theory, it is believed that ionic groups,such as a carboxylate group on a first dalbavancin monomer, aid in thestabilization of dalbavancin multimers by forming ionic interactionswith oppositely charged ions, such as tertiary nitrogen groups, on asecond dalbavancin monomer. Such ionic interactions can be influenced bypH. It is believed that the increasing tendency of dalbavancin to bepresent as a multimer at higher pH is indication that ionic interactionsare important in multimer stabilization. In particular, it is believedthat dalbavancin multimers are destabilized at lower pH, presumably dueto interruption of the ionic interactions contributing to multimerstability as certain functional groups, such as carboxylate groups, maybe protonated at lower pH values.

Influence of Solution Ionic Strength on the Population Ratio ofDalbavancin Multimer to Monomer

The influence of solution ionic strength on the population ratio ofdalbavancin multimer to monomer was determined by mass spectrometry. Themass spectra were obtained in electrospray positive mode on a FinniganLCQ^(Deca) ion trap instrument previously tuned and calibrated inelectrospray mode using Ultramark 1621, caffeine and MRFA(L-metionyl-arginyl-phenilalanyl-arginine). All the mass spectra wererecorded using the conditions listed in Table 13. The sample parametersthat were investigated are listed in Table 14.

TABLE 13 MS Conditions Sample Inlet Conditions: Capillary Temperature (°C.) 200 Sheat Gas (N₂ arbitrary units): 40 Sample Inlet VoltageSettings: Polarity: positive Source Voltage (kV): 4.70 Capillary Voltage(V): 34 Tube Lens Offset (V): −60 Full Scan conditions: Scan range(amu):  500-2000 Number of microscans: 3 Maximum ion time (ms): 200 ZoomScan conditions: Scan range (amu): 1218-1228 Number of microscans: 5Maximum ion time (ms): 50

TABLE 14 Sample Parameters Sample μg/mL Solvent pH 100 COO—NH₄ ⁺ 5 mM 5Dalbavancin 100 COO—NH₄ ⁺ 50 mM 5 100 COO—NH₄ ⁺ 100 mM 5

Sample water solutions were infused at 10 μL/min via a Harward syringepump and the mass spectra were obtained as seen in FIGS. 6-8.

The obtained spectra indicate that the population ratio of dalbavancinmultimer to monomer is influenced by ionic strength. An increase inbuffer concentration was found to correspond to a decrease in multimermass traces and hence a decrease in dalbavancin multimer to monomerpopulation ratio.

As mentioned, it is believed that ionic interactions are important indalbavancin multimer stability. The fact that increasing ionic strengthwas correlated with decreasing intensity of multimer mass tracessubstantiates the role of ionic interactions in multimer stability.However, as multimer mass traces were present even at higher ionicstrengths, another, second, interaction may be involved in multimerstabilization.

While not bound to any theory, it is believed that hydrophobicinteractions are important in stabilizing the multimer species ofdalbavancin. If the stabilization of these non-covalent dalbavancinmultimers was solely due to ionic interactions, it would be expectedthat an increase in ionic strength would result in total loss ofmultimer mass species. That is, it would be expected that as the ionicstrength of the solution increases, the ionic interactions stabilizingthe multimer would be disrupted by the increased population of ions insolution, with which the monomers would more readily associate.Consequently, the solution ionic strength would drive the multimers todisassociate into monomer components and the resulting mass spectrawould be free of any multimer mass traces. However, even at highsolution ionic strength (such as 100 mM ammonium formate), the presenceof dalbavancin multimers is detected in the mass spectrum. Accordingly,the multimers of dalbavancin are deemed to be stabilized, at least inpart, by hydrophobic interactions.

Structurally Similar Compound

It is believed that the improved efficacy of Dalbavancin is due at leastin part to its ability to form multimers. It is thought that this uniquecharacteristic is not shared even by very structurally similarcompounds. A compound with a chemical structure similar to Dalbavancinwas investigated by mass spectroscopy analysis for its ability to formmultimers. The mass spectra were obtained in electrospray positive modeon a Finnigan LCQ^(Deca) ion trap instrument previous tuned andcalibrated in electrospray mode using Ultramark 1621, caffeine and MRFA(L-metionyl-arginyl-phenilalanyl-arginine). All the mass spectra wererecorded using the conditions listed in Table 15. The sample parametersthat were investigated are listed in Table 16. Sample water solutionswere infused at 10 μL/min via a Harward syringe pump and mass spectrawere obtained as seen in FIGS. 9 and 10.

TABLE 15 Mass Spectra Conditions Sample Inlet Conditions: CapillaryTemperature (° C.) 200 Sheat Gas (N₂ arbitrary units): 40 Sample InletVoltage Settings: Polarity: positive Source Voltage (kV): 4.70 CapillaryVoltage (V): 34 Tube Lens Offset (V): −60 Full Scan conditions: Scanrange (amu):  500-2000 Number of microscans: 3 Maximum ion time (ms):200 Zoom Scan conditions: Scan range (amu): 1218-1228 Number ofmicroscans: 5 Maximum ion time (ms): 50

TABLE 16 Sample Parameters Sample μg/mL Solvent pH Teicoplanin 50 H₂On.a 100 H₂O n.a. n.a. = not adjusted

A similar glycopeptide antibiotic (teicoplanin) does not show multimericcomplexes in solution at various concentrations. This supports theindication that structurally similar compounds fail to form multimericspecies in solution, and that this phenomenon may play an important rolein the activity of the dalbavancin.

Example 7 Matrix-Assisted Laser Desorption/Ionisation Time of Flight(MALDI-TOE) Mass Spectrometry of Protein-Dalbavancin Complexes

10 ul HSA, 0.150 mM was mixed with 10 ul dalbavancin solution (from0.075 mM, 0.15 mM, 0.3 mM and 1.5 mM) and incubated for 60 min at 37° C.The samples were prepared for analysis using the dried droplettechnique. Spectra were obtained on a BRUKER FLEX III, tof massspectrometer previously tuned and calibrated using standard bovine serumalbumin, acquiring and averaging spectra generated by 200 laser shots.Matrix: 9 parts of DHB-9 (2,5-dihydroxy-benzoic acid) saturated inacetonitrile/H₂O (50:50), 1 part of sinapinic acid saturated inacetonitrile/H₂O (50:50). 0.5 ul of sample solution and 0.5 ul of matrixsolution were mixed and placed on the laser target.

Dalbavancin binds to the protein as the monomer (1 HAS+1 dalbavancin).At very high dalbavancin to protein ratios (1:2, 1:10), the presence ofcomplexes containing 2 molecules of dalbavancin per protein molecule canbe observed.

Example 8 Isothermal Titration Calorimetry of Binding of Dalbavancin toN,N′-diacetyl-Lys-D-Ala-D-Ala in the presence of Human Serum Albumin

The binding of dalbavancin to N,N′-diacetyl-Lys-D-Ala-D-Ala, a peptideanalog of cell-wall targets of dalbavancin, was investigated byisothermal titration calorimetry (ITC) in the presence of HSA over arange of concentrations (up to 600 μM) at 25° C., with some additionalmeasurements at 37° C. HSA increased the solubility of dalbavancin andreduced its binding affinity for the tri-peptide ligand. Results werecompared with those for vancomycin. The observed effects plateaued atrelatively low HSA concentrations, consistent with a non-competitivebinding model that allows binding of ligand to dalbavancin both free insolution and (more weakly) to the dalbavancin-HSA complex.

Preliminary experiments demonstrated that, in the absence of serumproteins, dalbavancin and vancomycin show similar binding profiles: bothgive exothermic binding to N,N′-diacetyl-Lys-D-Ala-D-Ala, but noevidence of binding to dipeptide (D-Ala-D-Ala) or toLys-D-Ala-D-Lactate. For dalbavancin/tri-peptide interaction, the datawere consistent with binding with K_(diss)=1-10 μM, depending ontemperature, similar to vancomycin under the same conditions. In thepresence of HSA, the solubility of dalbavancin is significantlyincreased and the binding affinity for tripeptide is reduced in a mannerconsistent with competitive or non-competitive binding by HSA for theantibiotic. The experiments described in this Example were designed inorder to: (a) compare dalbavancin/tri-peptide measurements at differenttemperatures (25 and 37° C.) and different HSA concentrations; (b) usethese data to construct a binding model to compare with observednumbers.

Dalbavancin was supplied by Biosearch Italia. Other reagents were fromSigma: vancomycin hydrochloride (Sigma V-2002, fw 1485.7),N,N′-diacetyl-Lys-D-Ala-D-Ala (Sigma D-9904, fw 372.4), human albumin(HSA; Sigma A-3782; mw 69,366).

Antibiotics and peptides were dissolved in aqueous buffer (20 mM Naphosphate, 150 mM NaCl, pH 7.4) containing HSA, with gentle stirringimmediately before each experiment. Peptide concentrations weredetermined by weight. Dalbavancin concentrations were determined eitherby weight or by UV absorbance using the molar extinction coefficientsε=12430 (dalbavancin, A₂₈₀ ^(1%)=68.42), ε₂₈₀=6690 (vancomycin). HSAconcentrations were determined by UV absorbance (HSA, ε₂₈₀=37,700; A₂₈₀^(1%)=5.44). Spectra were recorded at room temperature in 1 cmpathlength quartz cuvettes using Shimadzu UV-160A or UV-1601spectrophotometers, with samples diluted quantitatively with buffer ifnecessary to give absorbance in the 0.1-1 A range.

Isothermal titration calorimetry was performed using a Microcal VP-ITCinstrument at 25° C. and 37° C. using standard operating procedures.See, e.g., Wiseman et al., Anal. Biochem. (1989) 179, 131-137; Cooper,et al., Philos. Trans. R. Soc. Lond. Ser. A-Math. Phys. Eng. Sci. (1993)345, 23-35; Cooper, A, Isothermal Titration Microcalorimetry in C.Jones, B. Mulloy and A. H. Thomas (Eds.), Microscopy, OpticalSpectroscopy, and Macroscopic Techniques. Humana Press, Totowa, N.J.,(1994) p 137-150; Cooper, A., Microcalorimetry of Protein-proteinInteractions in J. E. Ladbury and B. Z. Chowdhry (Eds.); Biocalorimetry:The Applications of Calorimetry in the Biological Sciences. Wiley,(1998) p 103-111; and Cooper, A., Curr. Opin. Chem. Biol. (1999) 3,557-563. Samples were degassed gently prior to loading to prevent bubbleformation in the calorimeter cell. Each experiment typically comprised25×10 μl injections of peptide solution (≈1 mM) into the calorimetercell (volume≈1.4 ml) containing antibiotic solution (≈20-100 μM).Control experiments involved injection of ligand into buffer underidentical conditions to determine heats of peptide dilution, and thesevalues were used for correction of raw binding data prior to analysis.Dalbavancin/tri-peptide binding experiments were repeated several timesat each temperature. ITC binding data were analysed using standardMicrocal ORIGIN™ software to determine the apparent number of bindingsites (N), the binding affinity (K_(ass)=1/K_(diss)) and enthalpy ofbinding (ΔH).

TABLE 17 Thermodynamic data for binding of tri-peptide binding todalbavancin and vancomycin determined by ITC assuming a simplenon-cooperative binding model: effects of temperature and HSA. K_(ass)ΔH ΔS T ° C. N M⁻¹ Kdiss μM Kcal/mol Eu [HSA] μM Dalbavancin 10 0.596.70E+05 1.49 −10.26 −9.60 0 10 0.59 9.20E+05 1.09 −8.95 −4.30 0 10 0.596.85E+05 1.46 −10.30 −9.60 0 10 0.56 6.24E+05 1.60 −10.30 −9.90 0 25 0.63.13E+05 3.19 −10.10 −8.90 0 25 x 3.30E+05 3.03 −11.30 −12.60 0 25 x3.20E+05 3.13 −11.70 −14.00 0 25 x 2.80E+05 3.57 −14.30 −23.00 0 25 0.573.03E+05 3.30 −12.6 −17.00 0 25 0.74 3.19E+05 3.13 −11.20 −12.50 0 withHSA 25 0.37 1.18E+05 8.47 −26.40 −65.50 13.6 with HSA 25 0.35 1.18E+058.47 −27.8 −70.10 13.6 with HSA 25 0.68 3.50E+04 28.57 −20.00 −46.2034.2 with HSA 25 0.83 2.76E+04 36.23 −16.90 −36.50 80.7 with HSA 25 1.383.49E+04 28.65 −18.60 −41.70 104 with HSA 25 0.9 3.10E+04 32.26 −21.05−50.00 288 with HSA 25 1.242 2.84E+04 35.21 −19.50 −44.9 430 with HSA 250.86 2.18E+04 45.87 −15.00 −30.40 601 37 0.82 1.30E+05 7.69 −12.50−16.80 0 37 0.6 1.10E+05 9.09 −17.4 −33.20 0 with HSA 37 0.179 1.25E+0480.00 −98.60 −299.00 482 with HSA 37 0.5 9568 104.52 −26.60 −67.60 516Vancomycin 10 1.1 6.90E+05 1.45 −9.49 −6.80 0 25 0.91 3.40E+05 2.94−10.70 −10.60 0 25 0.97 3.60E+05 2.78 −12.90 −17.80 0 with HSA 25 1.052.90E+05 3.45 −10.09 −8.80 513

ITC experiments on the binding of tri-peptide to dalbavancin in theabsence of HSA give consistent data for binding affinities, with averageK_(diss) in the region of 1.4, 3. 1, and 8.4 μM at 10, 25, and 37° C.respectively (Table 17). Binding is exothermic. Concentrationcalculations here indicate that N is closer to 0.5 under theseconditions. This is consistent with the binding of one tri-peptidemolecule per dalbavancin dimer under these conditions. These bindingaffinities and enthalpies of binding are comparable to those observedwith vancomycin under the same conditions (Table 17, and D. McPhail, A.Cooper, J. Chem. Soc.—Faraday Trans. (1997) 93, 2283-2289.). Note alsothat vancomycin undergoes ligand-induced dimerization at higherconcentrations.

Addition of HSA to the dalbavancin mixtures reduces the apparent bindingaffinity between tri-peptide and dalbavancin, though the binding isapparently more exothermic (Table 17). The HSA concentration dependencefor this at 25° C. is shown in FIG. 11. After an initial rise(weakening) in apparent K_(diss) up to 35 μM HSA, it remains relativelyconstant for higher HSA concentrations approaching physiological levels(600 μM). The plateau level at high concentrations of HSA (K_(diss)≈35μM) corresponds to around 10-12× weaker binding affinity than in theabsence of HSA. A similar reduction is seen at 37° C.

The HSA effect was not due to interaction with the tri-peptide. ControlITC experiments for binding of vancomycin to tri-peptide in the presenceof HSA gave results comparable to those seen in the absence of HSA (seeTable 17). This indicates that neither the peptide nor the closelyrelated antibiotic, vancomycin, interact with HSA in solution. Itfollows that any effect of HSA on dalbavancin/tri-peptide interactionmust be due to interaction between HSA and dalbavancin.

Although not wishing to be bound by theory, the above data areconsistent with a non-competitive binding model. This model assumes thatthe tri-peptide ligand (L) can bind to dalbavancin (D) both in the freestate and (possibly with different affinity) in the dalbavancin-HSAcomplex.

D+L⇄DL; K _(L) =[D][L]/[DL]

D+HSA⇄D.HSA; K _(HSA) =[D][HSA]/[D.HSA]

D.HSA+L⇄LD.HSA; K _(LDSA) =[D.HSA][L]/[LD.HSA]

Apparent (Observed) Ligand Binding Dissociation Constant(Non-Competitive):

$\begin{matrix}{K_{{app},L} = \frac{\left\lbrack {{total}{\mspace{11mu} \;}D} \right\rbrack \lbrack L\rbrack}{\left\lbrack {{total}\mspace{14mu} {DL}\mspace{14mu} {complex}} \right\rbrack}} \\{= \frac{\left( {\lbrack D\rbrack + \left\lbrack {D \cdot {HSA}} \right\rbrack} \right)\lbrack L\rbrack}{\left( {\lbrack{DL}\rbrack + \left\lbrack {{LD} \cdot {HSA}} \right\rbrack} \right)}} \\{= {K_{L}{\left\{ \frac{1 + \lbrack{HSA}\rbrack}{K_{HSA}} \right\}/\left\{ \frac{1 + {\lbrack{HSA}\rbrack \cdot K_{L}}}{K_{HSA} \cdot K_{LDHSA}} \right\}}}}\end{matrix}$

This shows a hyperbolic dependence of K_(app,L) on free HSAconcentration that agrees well with the observed data (FIG. 11). At high[HSA] this reaches an asymptotic (plateau) value

K _(app,L) =K _(LDHSA)(for large [HSA])

This suggests that the binding affinity for tri-peptide is around 35 μMwhen dalbavancin is bound to HSA, compared to 3 μM for free dalbavancin(25° C. figures).

Further mechanisms may be suggested in terms of whether dalbavancin isacting as a monomer or a dimer in its interactions with peptides orproteins. Direct comparison of dalbavancin with vancomycin (which showsunambiguous 1:1 binding at these low concentrations) shows that bindingis complete at lower molar ratios (lower N) for dalbavancin (FIG. 12).This is consistent with a 2:1 dalbavancin:peptide complex.

However, in the presence of HSA, the apparent N values increase (Table17), and may be more consistent with 1:1 complexation. Although notwishing to be bound by theory, the model shown in FIG. 13, showing thepossible interaction of dalbavancin monomers and dimers with tri-peptideligands and HSA, is consistent with these observations. FIG. 13A depictsdalbavancin as in monomer-dimer equilibrium in solution (predominantlyas dimer), but binding as monomer to two separate sites on HSA. This isconsistent with the N=0.5 values observed by ITC for binding of serumproteins to dalbavancin (Example 3). FIG. 13B depicts ligand binding tothe dalbavancin dimer in solution, and (more weakly) to the dalbavancinmonomers attached to HSA. This is consistent with the non-competitivebinding of dalbavancin to both tri-peptide and to HSA, with variableapparent stoichiometries.

In sum, this Example shows that HSA reduces the binding affinity ofdalbavancin for tri-peptide ligand in a manner consistent with anon-competitive mechanism and that dalbavancin bound to HSA retains itsability to bind tri-peptide ligands, albeit with reduced affinity. Theseresults are also consistent with a model in which dalbavancin is inmonomer-multimer equilibrium, predominantly multimeric in solution, withstrong affinity of multimers for peptide ligand. Dalbavancin monomers,both free and bound to serum albumin, may also bind peptides with areduced affinity.

Example 9 A-40926 and Dalbavancin Preparations Preparation of A-40926

A-40926, the natural glycopeptide produced by fermentation, was thestarting material for producing Dalbavancin. It was produced by aNonomuria sp ATCC 39727 as a mixture of A-40926 and its acetylderivative (see U.S. Pat. No. 4,935,238 and B. Golstain et al.Antimicrobial Agent and Chemotherapy, December 1987, p. 1961-1966). Theacetyl derivative was first deacetylated to A-40926. Afterdeacetylation, A-40926 was purified by column chromatography onpolyamide as described below. The following description isrepresentative of the current production method. The amounts reportedhere are about ¼ of the amounts usually worked in an industrialpreparation.

Deacylation of A-40926

10 m³ of fermentation broth (23° C.) containing a total of about 1 g/Lof A-40926 and its acetyl derivative were adjusted, under stirring, atpH 11.4 with NaOH 30%. Stirring was continued for 6 hours, then thetemperature was reduced to 15° C. and the broth was microfiltered (KochProtosep IV Microfilter with a 0.12 m² ceramic membrane 0.1). Duringmicrofiltration, water was continuously added to the retentate in orderto obtain, at the end of the process, a permeate of 20-25 m³ and aretentate of 4.5-5 m³(one half of the starting value).

The permeate, which contained A-40926, was analyzed by HPLC. When thedeacetylation was completed, the pH of the permeate solution wasadjusted at pH 7 with 30% sulfuric acid (stored at 20° C.). In thisexample, 25 m³ of filtered broth containing 6.62 Kg of A-40926 (268mg/L) was obtained. The deacetylation yield was 66.2%. If themicrofiltration process was carried out for longer time and higherextraction volume than those employed in this process, the yield can beincreased up to 90%.

Purification of A-40926 on Polyamide Column

After extraction, A-40926 contained in filtered broth was purified on apolyamide column, as described below. The amount reported in thisdescription is about 1/10 of the amount usually worked in an industrialpreparation and is representative of the current production method.

500 L of polyamide resin SC6 (from Macherey Nagel) were suspended indemineralized water and loaded into a column. The resin was thenconditioned at pH 6-6.5 by eluting the column with at least 2 BV (bedvolume) of a buffer solution prepared by dissolving 4 Kg of sodiumcarbonate in 800 L of water and adjusting the pH of the resultedsolution with acetic acid.

The A-40926 filtered broth (9000 L; assay 0.275 mg/L; A-40926 2475 g; pH6±0.2; temperature 10±3° C.) was loaded into the column at about 5 g ofactivity per liter of resin (activity/resin ratio of 5-8 g/L was usuallyused). The column was washed with the following solutions: 3 BV (1500 L)of a solution at pH 6 prepared by dissolving 7.5 Kg of sodium carbonatein 1500 L of demineralized water and adjusting the pH with acetic acid;4 BV (2000 L) of a solution at pH 8 prepared by dissolving 10 Kg ofsodium carbonate in 2000 L of demineralized water and adjusting the pHwith acetic acid; 1.5 BV (750 L) of a solution at pH 9 prepared bydissolving 4 Kg of sodium carbonate in 750 L of demineralized water andadjusting the pH with acetic acid.

A-40926 was recovered from the column by eluting with 4 BV (2000 L) of abuffer solution at pH 10 prepared by dissolving 10 Kg of sodiumcarbonate in 2000 L of demineralized water and adjusting the pH withacetic acid. The fractions containing purified A-40926 (concentration ofA-40926 greater than 0.5 g/L and HPLC area % of the main component(Bo+B₁) greater than 80%) were collected, neutralized with 1N HCl, andanalyzed by HPLC. About 2000 L of final clear solution were obtained.

The resin used for the purification was regenerated with 1.5 BV of 1:1mixture of isopropanol/5% NaOH followed by a washing with 5 BV ofdemineralized water.

A-40926 Concentration

The solution coming from the column was subject to several rounds ofdilution/concentration steps to eliminate most of the inorganic salts inthe solution. The solution was concentrated to 80 L by nanofiltrationusing a membrane with a cut-off of 250 D, diluted with 80 L ofdemineralized water, and re-concentrated at the starting volume (80 L)by nanofiltration. This operation was repeated at least 5 times. The pHof the final solution (80 L, pH 7.5) was adjusted at pH 6.3 with 23%HCl. The solution was then diluted with 80 L of acetone, and its pH wasadjusted again at pH 2.6 with 23% HCl.

Decoloring

680 g of charcoal PA 200 C (˜0.3 g/g A-40926) was added under stirringto the solution obtained in the above step (160 L). Stirring wascontinued for at least 30 minutes at room temperature, then about0.5-0.6 Kg of filter aid (DIF-BO) was added. The mixture was filteredthrough a filter cartridge. The clear solution obtained was concentratedunder vacuo (45° C.) in order to reduce the acetone below 10%. The finalvolume was about 100 L. The pH was then adjusted at 6.7 with aq. NaOH,and the concentration step was continued using the usual nanofilteruntil the A-40926 concentration was around 100 g/L. 20 L concentratedsolution was obtained (A-40926 1884 g, 94.2 g/L).

Precipitation and Drying

The previous solution was diluted under stirring with 20 L of acetone,and its pH was adjusted at 5.1 with 10% HCl. To this solution additional5 volumes of acetone (100 L) were added to complete the A-40926precipitation. If water content was not <15% at this point, additionalacetone was added. After 2 hours the suspension was centrifuged, and thesolid was washed with 3×10 L of fresh acetone. The mother liquors wereanalyzed and discharged after having ascertained the absence of product.

Solid A-40926 was dried under reduced pressure at 30-35° C. in a staticdrier until the residual acetone was below 2% and the water was lessthan 10%. The product was then sieved through a 50 mesh sieve obtaining2.08 Kg of purified A-40926 (HPLC assay 81.4%; water 6.2%; sulphatedashes 4.8%). The yield, starting from the activity loaded on the column,was 68.4%.

Synthesis of Dalbavancin

Dalbavancin (BI-397) was prepared from the natural glycopeptide A-40926through a three-step synthesis as described in Malabarba and Donadio(1999), Drugs of the Future, 24(8):839-846. Specifically, A-40926 wasfirst subject to an esterification step to make MA, which was thensubject to an amidation step to make MA-A-1. A final hydrolysis stepthen converted MA-A-I into dalbavancin.

Esterification step (Step 1)

The following description is representative of the current method inuse.

Preparation of H₂SO₄ 96%/MeOH (Solution A)

In a 15 L round bottomed flask equipped with a mechanical stirrer and athermometer, 2.28 L of H₂SO₄ 96% (˜300 mL of H₂SO₄ 96% per Kg of A-40926powder) was added drop wise to 7.9 L of MeOH. An external ice bath wasused to maintain the temperature between 0 and 5° C.

Reaction Procedure

Starting material A-40926 (7.6 kg; batch 019, assay 85.09%; activity6.46 kg; 3.73 mol) was suspended in a 140 L glass-lined reactor in MeOH(46 L), and the resulting suspension was cooled at 0° C. ±2° C. At thistemperature the suspension was treated with the previously preparedSolution A (H₂SO₄/MeOH). The resulting solution was stirred at 0° C. for22-26 hours while the reaction (a reaction aliquot diluted 100 timeswith 1:1 acetonitrile/water mixture) was monitored by HPLC analysisevery two hours. The esterification was considered complete when theresidual A-40926 was less than 5% and diester was not more than 10% asHPLC area %.

Ester (MA) Isolation

When the reaction was complete, the mixture was cooled at −5° C. (+/−2°C.) and diluted with a same volume of cold water (54 L) maintaining thetemperature below 5° C. The product (MA) was precipitated by adjustingthe pH of the solution at 5.5 (+/−0.2) by slowly adding 10.2 L oftriethylamine (TEA). Stirring was continued for an additional hour at0-2° C., then the solid obtained was centrifuged, washed with water (10L per Kg of A-40926) and finally with MeOH (3 L of MeOH per Kg ofstarting A-40926) previously cooled at 10-15° C. Washing with water wasdone primarily to remove sulphates from MA.

Mother liquors and washings were separately analyzed and discharged ifcontained less than 1-2% of activity. The product was dried in vacuo (50mmHg) at 35-40° C. (external temperature) until the residual water wasless than 10%. 7.6 Kg of MA (5.63 kg activity, 3.23 mol) was obtained asa brownish powder.

The analysis showed the following values of HPLC area %: MA 89.8,A-40926 3.2, Diester derivative 5.9. The HPLC assay was 74.2%, activity5.637 Kg; 3.23 mol; yield=86.5%. This material was used in the followingstep without any further purification.

Amidation Step (Step 2)

The following description is representative of the current productionmethod.

Preparation of the DMSO/HCl Mixture (Solution B)

DMSO (1.6 L) was placed into a 10 L round bottomed flask, equipped witha mechanical stirrer and a thermometer, and cooled with an ice bathbelow 10° C. HCl 37% (1 L) was then slowly added under stirringmaintaining the temperature of the mixture below 25° C.

Amidation Procedure (Production of MA-A-1)

Starting material MA 5.95 kg (assay 76.3%, KF 8.9%; 2.68 mol) was slowlydissolved under stirring in 19.2 L of 1:1 DMSO/MeOH mixture (^(˜)1.6 LDMSO and 1.6 L MeOH per Kg of MA powder) at room temperature. After 1hour of stirring 709 mL of 3-(dimethylamino)-propylamine (DMEPA, MW102.1; density=0.812 g/mL; 5.63 mol; 1.96 mols per mol of starting MA)and 325 g of 1-hydroxybenzotriazole hydrate (HOBTH₂O; MW 153.1; 2.04mol; 0.71 mol per mol of starting MA) were added to the reactionmixture. Stirring was continued until a complete solution was obtained,then the mixture was adjusted at pH 3-3.1 (measured after diluting analiquot of the reaction 10 times with water) by slowly adding about 2.0L of Solution B (DMSO/HCl).

A dicyclohexylcarbodiammide (DCC) solution, prepared by dissolving 1.03Kg of DCC (4.99 mol; MW 206.3; 1.74 mol per mol of MA) in 4.1 L of 1:1DMSO/MeOH mixture, was added to the stirred reaction mixture in 10minutes. Stirring was continued for 5 hours, then additional 51.5 g ofsolid DCC (0.25 mol) was added to the reaction mixture in order to lowerthe residual MA under 5%, maintaining the level of isoureas lower than4-5%. Isoureas are a group of by-products produced by further reactionof Dalbavancin with the excess of DCC.

Typically after 2 additional hours (7 hours total) the reaction wascompleted. At the end the mixture was diluted with water (60 L) to lowerthe DMSO concentration to 15% (v/v) and adjusted at pH 2.3 with HCl 1N(0.85 L) to destroy any residual DCC.

Hydrolysis of MA-A-I to Dalbavancin (Step 3)

After 30 minutes the mixture was adjusted at pH 12.0-12.1 with 15% NaOH(8 L). Stirring was continued for 4 hours maintaining the mixture atthis pH with small additions of NaOH 15%. After this time the residualMA-A-1 was less than 0.2% as HPLC area %.

The mixture was then acidified at pH 3.0 with 1N HCl (19 L), and thesuspension was filtered to remove the dicyclohexylurea formed. The solidcake was washed on the filter with demineralized water (2×20 L).Washings and filtrate were gathered together, obtaining a clear solutionwhich was analyzed by HPLC. 152.8 L of solution containing 21.74 g/L ofDalbavancin (total activity 3322 g; 1.828 mol, yield=68.2%) wasobtained.

Purification of Dalbavancin

The following description is representative of the current productionmethod.

The 152.8 L of solution obtained from the hydrolysis step and containing3322 g of Dalbavancin activity was split into two parts and each one waspurified separately on the same chromatographic column containing 400 Lof polyamide. In these two purification runs the activity/resin ratiowere 4.3 and 4.0 g/L respectively.

Polyamide Column Preparation

The glass-lined column (internal diameter=40 cm, h=320 cm) containing400 L of polyamide resin was cleaned according to the resin regenerationprocedures (see below) and conditioned with 2 BV (800 L) ofdemineralized water acidified with 4 L of AcOH (pH=3.2).

Purification of the First Portion

The first portion of 76.4 L of starting solution was diluted with H₂O(56 L) in order to lower the DMSO content under 5% (v/v), and acidifiedto pH 2.78 with 1 N HCl (3.4 L). This solution was then loaded onto thecolumn at a flow rate of 150 L/h. After loading, the resin was washedwith the following solutions: 4 BV (1600 L) of H₂O acidified with AcOH(8 L), pH=3.2; 5 BV (2000 L) of AcONa 0.1 M, pH=8.2; 1 BV (400 L) of H₂Oacidified with AcOH (1 L), pH=3.2. Dalbavancin was eluted with 4 BV(2400 L) of H₂O/MeOH (8:2) acidified with AcOH (6 L), pH=3.4.

During the elution step, 22 fractions of 50-60 L each were collected andanalyzed by HPLC. Fractions from 9 to 25 (concentration of Dalbavancinhigher than 0.5 g/L and HPLC area % of (B₀+B₁)>80%) were pooledtogether, obtaining 969 L of solution with 1.56 Kg of Dalbavancin(yield=93.9%). This solution was then concentrated by nanofiltration,obtaining 125.7 L of solution with 1.38 Kg of Dalbavancin. 850 L ofpermeate, containing 145 g of impure Dalbavancin (8.7%), wereneutralized and discharged.

Resin Regeneration

Before re-using the resin was washed with the following solutions: 2.5BV (1000 L) of 1:1 MeOH/water acidified with acetic acid (2.5 mL/L); 2.5BV (1000 L) of 1:1 0.5% NaOH/isopropanol; 10 BV (4000 L) ofdemineralized water. The resin was then re-equilibrated with BV (800 L)of water acidified with acetic acid (2.5 mL/L).

Purification of the Second Portion

The second portion of the starting solution coming from the hydrolysisstep (76.5 L) was diluted with H₂O (56 L) to lower the DMSO contentunder 5% (v/v) and acidified to pH 2.87 with 3.0 L of 1 N HCl. Theportion was then purified as previously described in the purification ofthe first portion. The pooled fractions (vol.=972 L, Dalbavancin 1.54Kg, yield=92.7%) were concentrated by nanofiltration, obtaining 133 L ofsolution with 1.46 Kg of activity. 850 L of permeate, containing 73 g ofDalbavancin (4.3%), was discharged.

The concentrated solutions coming from the two purification steps werere-analyzed and pooled together giving 258 L of solution containing 2840g of purified Dalbavancin. The purification yield was 86%. The totalyield, starting from MA, was 58.3%.

Final Polyamide Regeneration

After the second purification run polyamide was regenerated with: 2.5 BVof 1:1 MeOH-water, acidified with AcOH (2.5 L) at pH=3.4; 2.5 BV of 1:10.5% NaOH-isopropanol; 10 BV of demineralized water.

Decoloration and Precipitation of Dalbavancin

1.5 mol of 1N HCl per mol of Dalbavancin and 0.3 g of charcoal CG1 (0.85Kg, from NORIT) per gram of Dalbavancin were added to the 258 L solutionobtained above. The mixture was stirred at room temperature for at least45 min. The pH was 3.1. The suspension was then filtered on a SUPRA DISCcartridge mod. SDP-EK1 from SEITZ-SCHENK, and the cake was washed with50 L of H₂O/MeOH 8:2. The filtrate was analyzed and concentrated againby nanofiltration, using a MPS 44 membrane with a cut off of 250 D. 21.3L of concentrated solution containing 119 g/L of Dalbavancin (pH 4.1;MeOH 1.9%, GC) was obtained. 909 mL of 1 N HCl was finally added toadjust the pH at 2.63, which corresponds to the salification ratio of1.65 molHcl/mol_(Dalbavancin).

The solution (22.2 L) was poured out, under stirring, in 200 L ofacetone. The solid obtained after decanting was centrifuged and washedwith 14 L of fresh acetone. The product was then dried under reducedpressure (50 mmHg) at 35° C., maintaining the internal temperature under30° C. for 17 hours. During the drying process, 1 L of pyrogen-freewater (<250 EU/mL), divided in two portions of 0.5 L each, was sprayedon the solid after three and five hours in order to remove the residualacetone that otherwise is difficult to eliminate. The product was thensieved (50 mesh), obtaining 2592 g of Dalbavancin (HPLC Assay 82.4%;water (KF) 14%; C_(1-3.0)%).

Example 10 Alternative Methods for A-40926 and Dalbavancin Preparation

The methods described below are alternative methods that can used in theA-40926 and Dalbavancin preparation process.

A-40926 Preparation on XAD-7 HP Deacetylation and MyceliumMicrofiltration

150 L of fermentation broth containing A-40926 (pH 7) were stirred in asuitable reactor at room temperature (24° C.), and adjusted at pH 11.5with a 2.5 N NaOH solution (2.5 L). After 4 hours of stirring, the brothwas adjusted at pH 10.6 with 15% HCl, and microfiltered through a 0.2micron membrane. 439 L of clear permeate was collected and thenconcentrated by nanofiltration using a MPS 44 membrane with a 250 D cutoff. The A-40926 concentrated solution obtained (58.6 L; 3.89 g/L) wasadjusted at pH 6.4 and stored at 4° C. until used.

Column Preparation and Purification.

XAD-7 HP resin (8 L) was suspended in a 1:1 water/methanol solution,filtered, and loaded into a proper glass-column (internal diameter 12cm) with a peristaltic pump.

The resin was then washed with water and equilibrated with 6 BV of asodium carbonate aqueous solution buffered at pH 6 prepared bydissolving 5 g of sodium carbonate per liter of water and adjusting thepH with acetic acid.

A portion of concentrated broth containing 194 g of A-40926 was loadedinto the XAD-7 HP column. The resin was then washed with the followingtwo buffered solutions, at a flow rate of 1/2 BV/hour, in order toeliminate part of the hydrophilic and the colored substances present: 3BV (24 L) of aq. 0.5% Acetic Acid solution adjusted at pH 5 with 30%sodium hydroxide; 5 BV (40 L) of a 8:2 mixture water/acetone with 5 mLof acetic acid/L of water.

A-40926 was finally eluted with 8 BV (64 L) of a 1:1 water/acetonemixture acidified with 5 mL of acetic acid/L of water. 16 fractions of 4L each were collected. The rich fractions (from 5 to 15) in whichA-40926 concentration was greater than 0.5 g/L were gathered togetherobtaining a solution containing 163.4 g of A-40926 (43 L, 3.8 g/L). Thecolumn yield was 81.3%. The other fractions (200 L) containing 0.23 g/L(45.3 g; 22.2%) of less pure A-40926 were discharged.

After the elution the resin was regenerated with 6 BV (55 L) of NaOH0.5%/isopropanol (1:1) mixture, and finally washed to neutral with 10 BVof water.

Charcoal Treatment.

The collected fractions were adjusted at pH 2.5 with HCl 37% (70 mL) andthen decolorized with 50 g charcoal type PA 200 (0.3 g/g of A-40926).The suspension obtained was stirred for 2 hours at room temperature andthen filtered through a KS 50 filter (d=25 cm, time=2.5 hours),obtaining 45.6 L of a slightly yellow A-40926 solution (3.5 g/L;yield=96.4%).

Concentration.

The decolorized solution was adjusted at pH 7 with NaOH 30% (230 mL) andconcentrated by nanofiltration and ultrafiltration. The use of thesetechniques was important for the elimination of the hydrophilicsubstances that were detected on the HPLC chromatograms at Rt=2-4minutes. When the retentate was concentrated to 1/10 of the startingvolume (4 L), the same volume of water was added and the solutionobtained was concentrated again. This concentration/dilution step wasrepeated three times in order to reduce the residual acetone to 0.25%.The final solution (2.2 L, 146.3 g of A-40926, 66.5 g/L, yield=91.5%)was analyzed by HPLC. The purification yield was 75.4%.

A-40926 Crystallization.

A 300 mL portion of the A-40926 solution (19.9 g of A-40926) was furtherconcentrated to 100 mL by using a laboratory scale ultrafilter and thenheated at 60-65° C. The pH of this solution was adjusted at 7 (30%NaOH), and 1.2 mL of 5:1 acetone/isopropanol mixture per mL ofconcentrated solution was added drop wise at this temperature. Theresulted mixture was left to cool at 20° C. After 1.5 hours, the solidobtained was filtered, washed on the filter with acetone, and dried at40° C. for 15 hours. 20.6 g of product (HPLC assay 82.0%; A-40926 16.9g) was obtained. The precipitation yield was 84.9%. The overall yield,starting from the filtered broth, was about 64%.

A-40926 Preparation on CG-71 Column Preparation

CG-71 resin (350 mL) was poured into a glass-column (internal diameter=4cm) and washed with water. The resin was equilibrated with 3 BV of asodium carbonate solution, prepared by dissolving 5 g of sodiumcarbonate in water at pH 6 with acetic acid. 250 mL fermentation broth(pH 7) containing 14.7 g of A-40926 was loaded into the column (42 g/Lresin). The resin was washed with the following three solutions: 1050 mL(3 BV) of aqueous solution of sodium carbonate (5 g/L) adjusted at pH 6with acetic acid; 1750 mL (5 BV) of aqueous solution of sodium carbonate(5 g/L) adjusted at pH 8 with acetic acid; 3150 mL (9 BV) of aqueoussolution of sodium carbonate (5 g/L) adjusted at pH 9 with acetic acid.

The activity was then eluted with 10 BV of demineralized water. 20fractions of 500 mL each were collected. Fractions 12 to 15 were pooledtogether, obtaining 2.2 L purified solution containing 11.7 g of A-40926(yield=79.6%). This solution was then concentrated by ultrafiltrationand the concentrate solution was further diluted with demineralizedwater and ultrafiltered again. The solution obtained was furtherconcentrated under reduced pressure to 50 mL.

A-40926 Crystallization.

The concentrated solution was heated at 60° C. and treated understirring with a 5:1 acetone/IPA mixture (60 mL). The mixture was thenslowly cooled at room temperature. The solid obtained was filtered,washed with acetone on the filter, and dried under vacuum at 35° C. for80 hours. 8.9 g of purified A-40926 (HPLC assay 84.2%) was obtained. Theoverall yield was 51%.

Alternative Amidation Step in Dalbavancin Synthesis UsingN-Methyl-2-Pyrrolidine (NMP) as a Solvent

MA mixture was added portion wise under stirring to a 1:1 NMP/MeOHmixture (64 mL). Stirring at 20-25° C. was continued until completesolution, then DMEPA (2.42 mL; 1.96 mol/eqMA) and HOBT (1.06 g; 0.71mol/eqMA) were added. The pH of the reaction mixture (checked on asample diluted 1:10 with water) was adjusted to 3.0 with 9.37 mL of 15%HCl in NMP (previously prepared from 34.0 mL HCl 37% dissolved in 57.7mL NMP). Then a solution of DCC (3.17 g; 1.57 mol/eqMA) in NMP/MeOH 1:1(12.7 mL) was added under stirring. The reaction was monitored by HPLC.The reaction was complete after about 6 hours (MA-A-1 88.9%, MA 7.3%,ISO 3.7%). This experiment suggests that NMP can be a convenientalternative to DMSO for the amidation reaction. The whole process wasnot influenced by this solvent change and the final Dalbavancin obtainedwas chemically equivalent to other batches.

Alternative Method of Dalbavancin Preparation: One-Pot Procedure

10 g of A-40926 complex (HPLC titer 80.66%, 4.6 mmole) was suspended in24 mL of MeOH under stirring at room temperature in a 100 mL glassreactor. The mixture was cooled at 0° C., and a solution of 4 g of HCl(g) in 16.4 mL of MeOH was added to complete the product solubilization.The temperature was then left to rise to 20° C. while stirring wascontinued for additional 24 hours.

After this time, 40 mL of DMSO and 0.4 g of HOBT were added to thereaction mixture.

1,1-dimethylamine propylamine was then added, adjusting the pH of theresulting reaction mixture between 3-3.1 (measured after diluting asample 9:1 with water). 1.8 g of solid DCC was then added and stirringwas continued for additional 15 hours. After this time the reactionmixture was transferred in a 1 L glass reactor and diluted with 80 mL ofwater. The pH was then brought to 12 by adding 240 mL of 15% NaOH.Stirring was continued for additional 60 minutes, and the mixture wasacidified at pH 2.8 with 260 mL of 15% aq. HCl. About 800 mL of finalclear solution containing 6.4 g of Dalbavancin was obtained (yield=76%).

HPLC analyses showed that the profile of the product obtained iscomparable with that obtained with the other manufacturing processes.

Although the foregoing invention has been described in some detail byway of illustration and examples for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced without departingfrom the spirit and scope of the invention. Therefore, the descriptionshould not be construed as limiting the scope of the invention, which isdelineated by the appended claims.

All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes andto the same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference.

1-64. (canceled)
 65. A pharmaceutical composition comprising:dalbavancin containing MAG in an amount of less than about 3 molepercent; at least one stabilizer, wherein said stabilizer inhibitsdegradation of one or more of the components of dalbavancin to lessactive or inactive materials; wherein the composition is lyophilized;and wherein the lyophilized pharmaceutical composition has a pH fromabout 3 to about 5 when reconstituted with water.
 66. The composition ofclaim 65, wherein the dalbavancin contains MAG in an amount of less thanabout 2.5 mole percent.
 67. The composition of claim 65, wherein thedalbavancin contains MAG in an amount of less than about 2 mole percent.68. The composition of claim 67, wherein the aqueous solution comprisingthe pharmaceutical composition has a pH from about 3.5 to 4.5.
 69. Thecomposition of claim 68, wherein the stabilizer comprises at least onesugar.
 70. The composition of claim 69, wherein the dalbavancin containsabout 85 to 96 mole percent of B₀.
 71. The composition of claim 65,wherein the dalbavancin contains MAG in an amount of less than about 1.5mole percent.